bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒08‒30
fifty-eight papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Transcriptome profiling analysis reveals that ATP6V0E2 is involved in the lysosomal activation by anlotinib.
  2. Lumenal Galectin-9-Lamp2 interaction regulates lysosome and autophagy to prevent pathogenesis in the intestine and pancreas.
  3. Loss of TMEM106B and PGRN leads to severe lysosomal abnormalities and neurodegeneration in mice.
  4. Role of induced pluripotent stem cells in lysosomal storage diseases.
  5. Ultrasensitive and specific two-photon fluorescence detection of hypochlorous acid by a lysosome-targeting fluorescent probe for cell imaging.
  6. Molecular insights into the human CLC-7/Ostm1 transporter.
  7. Arylsulfatase K inactivation causes mucopolysaccharidosis due to deficient glucuronate desulfation of heparan and chondroitin sulfate.
  8. In vitro and in vivo studies on potentiation of curcumin-induced lysosomal-dependent apoptosis upon silencing of cathepsin C in colorectal cancer cells.
  9. User guide to MiT-TFE isoforms and post-translational modifications.
  10. Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria.
  11. In vitro Validation of Chimeric β-Galactosylceramidase Enzymes With Improved Enzymatic Activity and Increased Secretion.
  12. The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis.
  13. Two-pore channels as master regulators of membrane trafficking and endocytic well-being.
  14. LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro.
  15. Clinicopathological Relationships in an Aged Case of DOORS Syndrome With a p.Arg506X Mutation in the ATP6V1B2 Gene.
  16. Diurnal Variation of Urinary Fabry Disease Biomarkers during Enzyme Replacement Therapy Cycles.
  17. Transcription Factor EB (TFEB): new metabolic coordinators mediating adaptive responses to exercise in skeletal muscle?
  18. ML-SA1, a selective TRPML agonist, inhibits DENV2 and ZIKV by promoting lysosomal acidification and protease activity.
  19. Downregulation of megalin, cubilin, ClC-5 and podocin in Fabry nephropathy: potential implications in the decreased effectiveness of enzyme replacement therapy.
  20. Loss of Two-Pore Channel 2 (TPC2) Expression Increases the Metastatic Traits of Melanoma Cells by a Mechanism Involving the Hippo Signalling Pathway and Store-Operated Calcium Entry.
  21. Patient with an autosomal-recessive MBTPS1-linked phenotype and clinical features of Silver-Russell syndrome.
  22. Cationic amphiphilic drugs induce elevation in lysoglycerophospholipid levels and cell death in leukemia cells.
  23. 1-Deoxysphingolipids cause autophagosome and lysosome accumulation and trigger NLRP3 inflammasome activation.
  24. Regulation of mTORC1 by Upstream Stimuli.
  25. Mechanism of Activation of Mechanistic Target of Rapamycin Complex 1 by Methionine.
  26. Searching for the real function of mTOR signaling in the regulation of PD-L1 expression.
  27. Serum- and glucocorticoid- inducible kinase 2, SGK2, is a novel autophagy regulator and modulates platinum drugs response in cancer cells.
  28. Dysregulation of metabolic flexibility: The impact of mTOR on autophagy in neurodegenerative disease.
  29. HGF protected against diabetic nephropathy via autophagy-lysosome pathway in podocyte by modulating PI3K/Akt-GSK3β-TFEB axis.
  30. GOLPH3 silencing inhibits adhesion of glioma U251 cells by regulating ITGB1 degradation under serum starvation.
  31. Rapamycin, by Inhibiting mTORC1 Signaling, Prevents the Loss of Striatal Bidirectional Synaptic Plasticity in a Rat Model of L-DOPA-Induced Dyskinesia.
  32. Prevention of Akt phosphorylation is a key to targeting cancer stem-like cells by mTOR inhibition.
  33. To cut or not to cut: New rules for proteolytic shedding of membrane proteins.
  34. Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection.
  35. Mitochondrial Regulation of the 26S Proteasome.
  36. The role of the mTOR pathway in models of drug-induced reward and the behavioural constituents of addiction.
  37. Expression of the Neuronal tRNA n-Tr20 Regulates Synaptic Transmission and Seizure Susceptibility.
  38. Defects in mRNA Translation in LRRK2-Mutant hiPSC-Derived Dopaminergic Neurons Lead to Dysregulated Calcium Homeostasis.
  39. The RUFYs, a Family of Effector Proteins Involved in Intracellular Trafficking and Cytoskeleton Dynamics.
  40. Structure of the C9orf72 ARF GAP complex that is haploinsufficient in ALS and FTD.
  41. Structure of human ATG9A: how holey art thou?
  42. Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy.
  43. Membrane Heterogeneity Controls Cellular Endocytic Trafficking.
  44. Lipophagy and Lipolysis Status in Lipid Storage and Lipid Metabolism Diseases.
  45. Mitophagy is induced by short ubiquitin chains on mitochondria.
  46. An insight of scientific developments in TSC for better therapeutic strategy.
  47. AMPK-Dependent Mechanisms but Not Hypothalamic Lipid Signaling Mediates GH-Secretory Responses to GHRH and Ghrelin.
  48. Regulatory T Cell Stability and Migration Are Dependent on mTOR.
  49. Sestrin2 Phosphorylation by ULK1 Induces Autophagic Degradation of Mitochondria Damaged by Copper-Induced Oxidative Stress.
  50. Identification of Annexin A2 as a key mTOR target to induce autophagy to autophagy dependent cell death in stress.
  51. Dysregulation of translational control signaling in autism spectrum disorders.
  52. Comprehensive Model for Epidermal Growth Factor Receptor Ligand Binding Involving Conformational States of the Extracellular and the Kinase Domains.
  53. Docosahexaenoic Acid, a Potential Treatment for Sarcopenia, Modulates the Ubiquitin-Proteasome and the Autophagy-Lysosome Systems.
  54. Chaperon-Mediated Autophagy Receptor Modulates Tumor Growth and Chemoresistance in Non-Small Cell Lung Cancer.
  55. An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy.
  56. Class II Arfs require a brefeldin-A-sensitive factor for Golgi association.
  57. TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals.
  58. The Endosomal Recycling Pathway-At the Crossroads of the Cell.
  1. Cell Death Dis. 2020 Aug 24. 11(8): 702
    Transcriptome profiling analysis reveals that ATP6V0E2 is involved in the lysosomal activation by anlotinib.
    Xin Sun, Yuhan Shu, Peiyi Yan, Hongliang Huang, Ruilan Gao, Mengting Xu, Liqin Lu, Jingkui Tian, Dongsheng Huang, Jianbin Zhang.
      Anlotinib is a receptor tyrosine kinase inhibitor with potential anti-neoplastic and anti-angiogenic activities. It has been approved for the treatment of non-small-cell lung cancer. Lysosomes are acidic organelles and have been implicated in various mechanisms of cancer therapeutics. However, the effect of anlotinib on lysosomal function has not been investigated. In the present study, anlotinib induces apoptosis in human colon cancer cells. Through transcriptome sequencing, we found for the first time that anlotinib treatment upregulates ATP6V0E2 (ATPase H+ Transporting V0 Subunit E2) and other lysosome-related genes expression in human colon cancer. In human colon cancer, we validated that anlotinib activates lysosomal function and enhances the fusion of autophagosomes and lysosomes. Moreover, anlotinib treatment is shown to inhibit mTOR (mammalian target of rapamycin) signaling and the activation of lysosomal function by anlotinib is mTOR dependent. Furthermore, anlotinib treatment activates TFEB, a key nuclear transcription factor that controls lysosome biogenesis and function. We found that anlotinib treatment promotes TFEB nuclear translocation and enhances its transcriptional activity. When TFEB or ATP6V0E2 are knocked down, the enhanced lysosomal function and autophagy by anlotinib are attenuated. Finally, inhibition of lysosomal function enhances anlotinib-induced cell death and tumor suppression, which may be attributed to high levels of ROS (reactive oxygen species). These findings suggest that the activation of lysosomal function protects against anlotinib-mediated cell apoptosis via regulating the cellular redox status. Taken together, our results provide novel insights into the regulatory mechanisms of anlotinib on lysosomes, and this information could facilitate the development of potential novel cancer therapeutic agents that inhibit lysosomal function.
    DOI:  https://doi.org/10.1038/s41419-020-02904-0
  2. Nat Commun. 2020 Aug 27. 11(1): 4286
    Lumenal Galectin-9-Lamp2 interaction regulates lysosome and autophagy to prevent pathogenesis in the intestine and pancreas.
    Janaki N Sudhakar, Hsueh-Han Lu, Hung-Yu Chiang, Ching-Shu Suen, Ming-Jing Hwang, Sung-Yu Wu, Chia-Ning Shen, Yao-Ming Chang, Fu-An Li, Fu-Tong Liu, Jr-Wen Shui.
      Intracellular galectins are carbohydrate-binding proteins capable of sensing and repairing damaged lysosomes. As in the physiological conditions glycosylated moieties are mostly in the lysosomal lumen but not cytosol, it is unclear whether galectins reside in lysosomes, bind to glycosylated proteins, and regulate lysosome functions. Here, we show in gut epithelial cells, galectin-9 is enriched in lysosomes and predominantly binds to lysosome-associated membrane protein 2 (Lamp2) in a Asn(N)-glycan dependent manner. At the steady state, galectin-9 binding to glycosylated Asn175 of Lamp2 is essential for functionality of lysosomes and autophagy. Loss of N-glycan-binding capability of galectin-9 causes its complete depletion from lysosomes and defective autophagy, leading to increased endoplasmic reticulum (ER) stress preferentially in autophagy-active Paneth cells and acinar cells. Unresolved ER stress consequently causes cell degeneration or apoptosis that associates with colitis and pancreatic disorders in mice. Therefore, lysosomal galectins maintain homeostatic function of lysosomes to prevent organ pathogenesis.
    DOI:  https://doi.org/10.1038/s41467-020-18102-7
  3. EMBO Rep. 2020 Aug 10. e50219
    Loss of TMEM106B and PGRN leads to severe lysosomal abnormalities and neurodegeneration in mice.
    Tuancheng Feng, Shuyi Mai, Jenn Marie Roscoe, Rory R Sheng, Mohammed Ullah, Junke Zhang, Isabel Iscol Katz, Haiyuan Yu, Wenjun Xiong, Fenghua Hu.
      Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). Loss of PGRN leads to lysosome dysfunction during aging. TMEM106B, a gene encoding a lysosomal membrane protein, is the main risk factor for FTLD with PGRN haploinsufficiency. But how TMEM106B affects FTLD disease progression remains to be determined. Here, we report that TMEM106B deficiency in mice leads to accumulation of lysosome vacuoles at the distal end of the axon initial segment in motor neurons and the development of FTLD-related pathology during aging. Ablation of both PGRN and TMEM106B in mice results in severe neuronal loss and glial activation in the spinal cord, retina, and brain. Enlarged lysosomes are frequently found in both microglia and astrocytes. Loss of both PGRN and TMEM106B results in an increased accumulation of lysosomal vacuoles in the axon initial segment of motor neurons and enhances the manifestation of FTLD phenotypes with a much earlier onset. These results provide novel insights into the role of TMEM106B in the lysosome, in brain aging, and in FTLD pathogenesis.
    Keywords:  TMEM106B; frontotemporal lobar degeneration; lysosome; neurodegeneration; progranulin
    DOI:  https://doi.org/10.15252/embr.202050219
  4. Mol Cell Neurosci. 2020 Aug 20. pii: S1044-7431(20)30163-9. [Epub ahead of print] 103540
    Role of induced pluripotent stem cells in lysosomal storage diseases.
    Jun Kido, Kimitoshi Nakamura, Takumi Era.
      Lysosomal storage diseases (LSDs) are a group of metabolism inborn errors caused by defective enzymes in the lysosome, resulting in the accumulation of undegraded substrates. Many characteristic cell features have been revealed in LSDs, including abnormal autophagy and mitochondrial dysfunction. The development of induced pluripotent stem cells (iPSCs) dramatically boosted research on LSDs, particularly regarding novel opportunities to clarify the disease etiology based on the storage of macromolecules, such as sphingolipids in lysosomes. iPSCs made from LSD patients (LSD-iPSCs) have been differentiated into neurons, endothelial cells, cardiomyocytes, hepatocytes, and macrophages, with each cell type closely resembling the primary disease phenotypes, providing new tools to probe the disease pathogenesis and to test therapeutic strategies. Abnormally accumulated substrates impaired autophagy and mitochondrial and synapse functions in LSD-iPSC-derived neurons. Reducing the accumulation with the treatment of drug candidates improved LSD-iPSC-derived neuron functions. Additionally, iPSC technology can help probe the gene expressions, proteomics, and metabolomics of LSDs. Further, gene repair and the generation of new mutations in causative genes in LSD-iPSCs can be used to understand both the specific roles of causative genes and the contributions of other genetic factors to these phenotypes. Moreover, the development of iPSC-derived organoids as disease models has bridged the gap between studies using cell lines and in vivo animal models. There are some reproducibility issues in iPSC research, however, including genetic and epigenetic abnormalities, such as chromosomal abnormalities, DNA mutations, and gene modifications via methylation. In this review, we present the disease and treatment concepts gathered using selected LSD-iPSCs, discuss iPSC research limitations, and set our future research visions. Such studies are expected to further inform and generate insights into LSDs and are important in research and clinical practice.
    Keywords:  Induced pluripotent stem cell; Lysosomal storage diseases; Neuron; Pathogenesis
    DOI:  https://doi.org/10.1016/j.mcn.2020.103540
  5. J Pharm Biomed Anal. 2020 Aug 13. pii: S0731-7085(20)31431-X. [Epub ahead of print]190 113545
    Ultrasensitive and specific two-photon fluorescence detection of hypochlorous acid by a lysosome-targeting fluorescent probe for cell imaging.
    Guan-Syuan Wu, Natesan Thirumalaivasan, Tsung-Chieh Lin, Shu-Pao Wu.
      Hypochlorous acid (HOCl) is involved in numerous cellular processes, such as pathogen response, immune regulation, and anti-inflammation. Consequently, the development of HOCl detection at the cellular level has been an important issue in investigating the dynamic distributions of HOCl. Herein, a fluorescent probe, Lyso-NA, containing a HOCl-reactive aminophenol group and a lysosomal-targeting morpholine group, has been effectively designed for detecting lysosomal HOCl. The reaction of Lyso-NA with HOCl induces the oxidation of aminophenol and accompanied by a 136-fold fluorescence enhancement. The detection limit is found at 13 nM. The fluorescence enhancement is accomplished through the suppression of twisted intramolecular charge transfer (TICT). With morpholine, the probe Lyso-NA shows the great lysosomal targetable ability for imaging endogenous lysosomal HOCl in living cells and tissues by two-photon microscopy, providing an opportunity to monitor HOCl in the lysosomes for understanding its biological functions.
    Keywords:  Fluorescent sensor; Hypochlorous acid; Lysosome; Two-photon effect
    DOI:  https://doi.org/10.1016/j.jpba.2020.113545
  6. Sci Adv. 2020 Aug;6(33): eabb4747
    Molecular insights into the human CLC-7/Ostm1 transporter.
    Sensen Zhang, Yang Liu, Bing Zhang, Jun Zhou, Tianyu Li, Zhiqiang Liu, Yang Li, Maojun Yang.
      CLC family proteins translocate chloride ions across cell membranes to maintain the membrane potential, regulate the transepithelial Cl- transport, and control the intravesicular pH among different organelles. CLC-7/Ostm1 is an electrogenic Cl-/H+ antiporter that mainly resides in lysosomes and osteoclast ruffled membranes. Mutations in human CLC-7/Ostm1 lead to lysosomal storage disorders and severe osteopetrosis. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human CLC-7/Ostm1 complex and reveal that the highly glycosylated Ostm1 functions like a lid positioned above CLC-7 and interacts extensively with CLC-7 within the membrane. Our complex structure reveals a functionally crucial domain interface between the amino terminus, TMD, and CBS domains of CLC-7. Structural analyses and electrophysiology studies suggest that the domain interaction interfaces affect the slow gating kinetics of CLC-7/Ostm1. Thus, our study deepens understanding of CLC-7/Ostm1 transporter and provides insights into the molecular basis of the disease-related mutations.
    DOI:  https://doi.org/10.1126/sciadv.abb4747
  7. Biochem J. 2020 Aug 28. pii: BCJ20200546. [Epub ahead of print]
    Arylsulfatase K inactivation causes mucopolysaccharidosis due to deficient glucuronate desulfation of heparan and chondroitin sulfate.
    Christof Trabszo, Bastian Ramms, Pradeep Chopra, Renate Lüllmann-Rauch, Stijn Stroobants, Jens Sproß, Anke Jeschke, Thorsten Schinke, Geert-Jan Boons, Jeffrey Esko, Torben Lübke, Thomas Dierks.
      Mucopolysaccharidoses comprise a group of rare metabolic diseases, in which the lysosomal degradation of glycosaminoglycans (GAGs) is impaired due to genetically inherited defects of lysosomal enzymes involved in GAG catabolism. The resulting intralysosomal accumulation of GAG-derived metabolites consequently manifests in neurological symptoms and also peripheral abnormalities in various tissues like liver, kidney, spleen and bone. As each GAG consists of differently sulfated disaccharide units, it needs a specific, but also partly overlapping set of lysosomal enzymes to accomplish their complete degradation. Recently, we identified and characterized the lysosomal enzyme arylsulfatase K (Arsk) exhibiting glucuronate-2-sulfatase activity as needed for the degradation of heparan sulfate, chondroitin sulfate and dermatan sulfate. In the present study, we investigated the physiological relevance of Arsk by means of a constitutive Arsk knockout mouse model. A complete lack of glucuronate desulfation was demonstrated by a specific enzyme activity assay. Arsk-deficient mice show, in an organ-specific manner, a moderate accumulation of heparan sulfate and chondroitin sulfate metabolites characterized by 2-O-sulfated glucuronate moieties at their non-reducing ends. Pathophysiological studies reflect a rather mild phenotype including behavioral changes. Interestingly, no prominent lysosomal storage pathology like bone abnormalities were detected. Our results from the Arsk mouse model suggest a new although mild form of MPS, which we designate MPS type IIB.
    Keywords:  desulfation; glycosaminoglycan degradation; lysosomal storage disorders; lysosomal sulfatases; mucopolysaccharidosis
    DOI:  https://doi.org/10.1042/BCJ20200546
  8. Pharmacol Res. 2020 Aug 21. pii: S1043-6618(20)31464-X. [Epub ahead of print] 105156
    In vitro and in vivo studies on potentiation of curcumin-induced lysosomal-dependent apoptosis upon silencing of cathepsin C in colorectal cancer cells.
    Tejinder Pal Khaket, Mahendra Pal Singh, Imran Khan, Sun Chul Kang.
      Cathepsins are lysosomal acid hydrolases that make crucial contributions to tumor progression through a variety of signaling mechanisms, including autophagy, cell survival, chemotherapeutic resistance, and metastasis. Herein, we report that cathepsin C (CTSC) silencing upregulates the anticancer potential of curcumin in colorectal cancer cells (CRCs) both in vitro and in athymic mice xenografts. Curcumin treatment enhances CTSC level in CRCs; however, CTSC silencing with subsequent curcumin treatment (sequential treatment) induces ER stress and autophagic dysregulation accompanied by lysosomal permeabilization and ROS generation. This lysosomal permeabilization triggered the cytosolic CTSB mediated BID-dependent mitochondrial membrane permeabilization and thereby caspase-dependent apoptosis. This phenotype can be rescued by CTSB inhibition and NAC, which further supported the involvement of ROS and CTSB in apoptosis following sequential treatment. Indeed, the sequential CTSC silencing and curcumin treatment also significantly curtailed tumor volume as well as ameliorated cytosolic cyt c and tBID protein levels in tumor tissues compared to those in control and individual treatments of CTSC targeting and on curcumin treatment in nude mice xenografts. The results reveal that CTSC controls the curcumin-induced cytotoxic insult through autophagy maintenance both in vitro and in athymic mice xenografts, thereby providing an insight into the role of CTSC in chemoprevention of CRCs.
    Keywords:  Apoptosis; Autophagy; Cathepsins; Endoplasmic reticulum stress
    DOI:  https://doi.org/10.1016/j.phrs.2020.105156
  9. Pigment Cell Melanoma Res. 2020 Aug 26.
    User guide to MiT-TFE isoforms and post-translational modifications.
    Hong Nhung Vu, Ramile Dilshat, Valerie Fock, Eiríkur Steingrímsson.
      The Microphthalmia-associated transcription factor (MITF) is at the core of melanocyte and melanoma fate specification. The related factors TFEB and TFE3 have been shown to be instrumental for transcriptional regulation of genes involved in lysosome biogenesis and autophagy, cellular processes important for mediating nutrition signals and recycling of cellular materials, in many cell types. The MITF, TFEB, TFE3 and TFEC proteins are highly related. They share many structural and functional features and are targeted by the same signaling pathways. However, the existence of several isoforms of each factor and the increasing number of residues shown to be post-translationally modified by various signaling pathways poses a difficulty in indexing amino acid residues in different isoforms across the different proteins. Here we provide a resource manual to cross-reference amino acids and post-translational modifications in all isoforms of the MiT-TFE family in humans, mice and zebrafish and summarize the protein accession numbers for each isoform of these factors in the different genomic databases. This will facilitate future studies on the signaling pathways that regulate different isoforms of the MiT-TFE transcription factor family.
    DOI:  https://doi.org/10.1111/pcmr.12922
  10. Cell Death Dis. 2020 Aug 26. 11(8): 708
    Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria.
    Kun Wang, Yujuan Zhan, Bonan Chen, Yuhua Lu, Ting Yin, Shikun Zhou, Weibin Zhang, Xiaodong Liu, Biaoyan Du, Xianli Wei, Jianyong Xiao.
      Cancer cells have developed chemoresistance and have improved their survival through the upregulation of autophagic mechanisms that protect mitochondrial function. Here, we report that the traditional Chinese anticancer agent tubeimoside I (Tub), which is a potent inhibitor of autophagy, can promote mitochondria-associated apoptosis in lung cancer cells. We found that Tub disrupted both mitochondrial and lysosomal pathways. One of its mechanisms was the induction of DRP1-mediated mitochondrial fragmentation. Another mechanism was the blocking of late-stage autophagic flux via impairment of lysosomal acidification through V-ATPase inhibition; this blocks the removal of dysfunctional mitochondria and results in reactive oxygen species (ROS) accumulation. Excessive ROS accumulation causes damage to lysosomal membranes and increases lysosomal membrane permeability, which leads to the leakage of cathepsin B. Finally, cathepsin B upregulates Bax-mediated mitochondrial outer membrane permeability and, subsequently, cytosolic cytochrome C-mediated caspase-dependent apoptosis. Thus, the cancer cell killing effect of Tub is enhanced through the formation of a positive feedback loop. The killing effect of Tub on lung cancer cells was verified in xenografted mice. In summary, Tub exerts a dual anticancer effect that involves the disruption of mitochondrial and lysosomal pathways and their interaction and, thereby, has a specific and enhanced killing effect on lung cancer cells.
    DOI:  https://doi.org/10.1038/s41419-020-02915-x
  11. Front Mol Biosci. 2020 ;7 167
    In vitro Validation of Chimeric β-Galactosylceramidase Enzymes With Improved Enzymatic Activity and Increased Secretion.
    Alessandra Ricca, Federica Cascino, Francesco Morena, Sabata Martino, Angela Gritti.
      Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disease (LSD) caused by inherited defects of the β-galactosylceramidase (GALC) gene. The infantile forms display a rapid and aggressive central and peripheral nervous system (CNS and PNS) dysfunction. No treatments are available for GLD patients. Effective gene therapy (GT) strategies for GLD require a safe and widespread delivery of the functional GALC enzyme to all affected tissues/organs, and particularly to the CNS. The use of chimeric lysosomal enzymes with increased secretion and enhanced transport across the blood-brain barrier (BBB) that boost the efficacy of GT approaches in pre-clinical models of similar neurodegenerative LSDs may benefit GLD as well. Here, we tested the safety and biological efficacy of chimeric GALC enzymes engineered to express an alternative signal peptide (iduronate-2-sulfatase - IDSsp) and the low-density lipoprotein receptor (LDLr)-binding domain from the Apolipoprotein E II (ApoE II) in GLD murine neural and hematopoietic stem/progenitor cells and progeny, which are relevant cells types in the context of in vivo and ex vivo GT platforms. We show that the lentiviral vector-mediated expression of the chimeric GALC enzymes is safe and leads to supranormal enzymatic activity in both neural and hematopoietic cells. The IDSsp.GALC shows enhanced expression and secretion in comparison to the unmodified GALC. The chimeric GALC enzymes produced by LV-transduced cells reduce intracellular galactosylceramide (GalCer) storage and effectively cross-correct GLD murine neurons and glial cells, indicating that the transgenic enzymes are delivered to lysosomes, efficiently secreted, and functional. Of note, the expression of LDLr and LDLr-related proteins in GLD neurons and glial cells supports the exploitation of this system to enhance the GALC supply in affected CNS cells and tissues. These in vitro studies support the use of chimeric GALC enzymes to develop novel and more effective GT approaches for GLD.
    Keywords:  chimeric GALC enzyme; gene therapy; globoid cell leukodystrophy; hematopoietic stem/progenitor cells; lentiviral vectors; neural stem/progenitor cells
    DOI:  https://doi.org/10.3389/fmolb.2020.00167
  12. J Lipid Res. 2020 Aug 26. pii: jlr.RA120000895. [Epub ahead of print]
    The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis.
    Natalie Bruiners, Noton Dutta, Valentina Guerrini, Hugh Salamon, Ken D Yamaguchi, Petros C Karakousis, Maria Laura Gennaro.
      The rise of drug-resistant tuberculosis (TB) poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each essential branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, AMPK- mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.
    Keywords:  Cholesterol/Trafficking; Immunology; Lipids; Macrophages / monocytes; Mycobacterium tuberculosis; Statins; mTORC1 regulation
    DOI:  https://doi.org/10.1194/jlr.RA120000895
  13. Curr Opin Physiol. 2020 Aug 14.
    Two-pore channels as master regulators of membrane trafficking and endocytic well-being.
    Kristin Vassileva, Mark Marsh, Sandip Patel.
      Two-pore channels (TPCs) are a ubiquitous class of Ca2+- and Na+-permeable ion channels expressed within the endo-lysosomal system. They have emerged as central regulators of a wide array of physiological processes intimately linked to information processing. In this short review, we highlight how molecular and chemical strategies have uncovered multiple roles for TPCs in regulating various aspects of endo-lysosomal trafficking associated with disease. We summarise advances in the identification of new small molecules to pharmacologically target TPCs for medical benefit. Lastly, we discuss possible underpinning molecular mechanism(s) that translate TPC-mediated ionic fluxes to function.
    DOI:  https://doi.org/10.1016/j.cophys.2020.08.002
  14. Int J Mol Sci. 2020 Aug 25. pii: E6110. [Epub ahead of print]21(17):
    LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro.
    Ineke D C Jansen, Wikky Tigchelaar-Gutter, Jolanda M A Hogervorst, Teun J de Vries, Paul Saftig, Vincent Everts.
      Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa β (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.
    Keywords:  LAMP-2; RANKL; osteoblasts; osteoclasts
    DOI:  https://doi.org/10.3390/ijms21176110
  15. Front Neurol. 2020 ;11 767
    Clinicopathological Relationships in an Aged Case of DOORS Syndrome With a p.Arg506X Mutation in the ATP6V1B2 Gene.
    Dénes Zádori, Levente Szalárdy, Zita Reisz, Gabor G Kovacs, Rita Maszlag-Török, Norbert F Ajeawung, László Vécsei, Philippe M Campeau, Péter Klivényi.
      DOORS [deafness, onychodystrophy, osteodystrophy, intellectual disability (mental retardation), and seizures] syndrome can be caused by mutations in the TBC1D24 and ATP6V1B2 genes, both of which are involved in endolysosomal function. Because of its extreme rarity, to date, no detailed neuropathological assessment has been performed to establish clinicopathological relationships and, thereby, understand better the neurobiology of this disease in aged cases. Accordingly, the aim of the current study was to highlight the clinicopathological characteristics of a novel case with a presumable de novo mutation in the ATP6V1B2 gene from a neuropathological point of view. This Caucasian male patient, who died at the age of 72 years, presented all the typical cardinal signs of DOORS syndrome. In addition, behavioral alterations, pyramidal signs, and Parkinsonism were observed. The p.R506X pathogenic mutation identified in the ATP6V1B2 gene was responsible for the clinical phenotype. The detailed neuropathological assessment revealed a limbic-predominant tauopathy in the forms of argyrophilic grain disease, primary age-related tauopathy, and age-related tau-astrogliopathy. In summary, we present the first detailed clinicopathological report of a patient with DOORS syndrome harboring a pathogenic mutation in the ATP6V1B2 gene. The demonstrated tauopathy may be considered as a consequence of lysosomal and/or mitochondrial dysfunction, similar to that found in Niemann-Pick type C disease, which is another lysosomal disorder characterized by premature neurodegenerative disorder.
    Keywords:  ATP6V1B2 gene; DOORS syndrome; lysosome; neuropathology; tauopathy
    DOI:  https://doi.org/10.3389/fneur.2020.00767
  16. Int J Mol Sci. 2020 Aug 25. pii: E6114. [Epub ahead of print]21(17):
    Diurnal Variation of Urinary Fabry Disease Biomarkers during Enzyme Replacement Therapy Cycles.
    Michel Boutin, Pamela Lavoie, Iskren Menkovic, Amanda Toupin, Mona Abaoui, Maha Elidrissi-Elawad, Marie-Françoise Arthus, Carole Fortier, Claudia Ménard, Bruno Maranda, Daniel G Bichet, Christiane Auray-Blais.
      Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the α-galactosidase A enzyme. This enzyme cleaves the last sugar unit of glycosphingolipids, including globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide (Ga2). Enzyme impairment leads to substrate accumulation in different organs, vascular endothelia, and biological fluids. Enzyme replacement therapy (ERT) is a commonly used treatment. Urinary analysis of Gb3 isoforms (different fatty acid moieties), as well as lyso-Gb3 and its analogues, is a reliable way to monitor treatment. These analogues correspond to lyso-Gb3 with chemical modifications on the sphingosine moiety (-C2H4, -C2H4+O, -H2, -H2+O, +O, +H2O2, and +H2O3). The effects of sample collection time on urinary biomarker levels between ERT cycles were not previously documented. The main objective of this project was to analyze the aforementioned biomarkers in urine samples from seven Fabry disease patients (three treated males, three treated females, and one ERT-naïve male) collected twice a day (morning and evening) for 42 days (three ERT cycles). Except for one participant, our results show that the biomarker levels were generally more elevated in the evening. However, there was less variability in samples collected in the morning. No cyclic variations in biomarker levels were observed between ERT infusions.
    Keywords:  Fabry disease; diurnal variation; globotriaosylceramide; globotriaosylsphingosine; glycosphingolipids; mass spectrometry
    DOI:  https://doi.org/10.3390/ijms21176114
  17. Am J Physiol Endocrinol Metab. 2020 Aug 24.
    Transcription Factor EB (TFEB): new metabolic coordinators mediating adaptive responses to exercise in skeletal muscle?
    Greg Robert Markby, Kei Sakamoto.
      In response to the increased energy demands of contractions, skeletal muscle adapts remarkably well through acutely regulating metabolic pathways to maintain energy balance and in the longer term by regulating metabolic reprogramming such as remodeling and expanding the mitochondrial network. This long-term adaptive response involves modulation of gene expression at least partly through the regulation of specific transcription factors and transcriptional coactivators. The AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor-γ co-activator 1a (PGC1a) pathway has long been known to orchestrate contraction-mediated adaptive responses, although AMPK-/PGC1a-independent pathways have also been proposed. Transcription factor EB (TFEB) and TFE3, known as important regulators of lysosomal biogenesis and autophagic processes, have emerged as new metabolic coordinators. The activity of TFEB/TFE3 is regulated through post-translational modifications (i.e. phosphorylation) and spatial organization. Under nutrient/energy stress, TFEB/TFE3 get dephosphorylated and translocate to the nucleus where they activate transcription of their target genes. It has recently been reported that exercise promotes nuclear translocation and activation of TFEB/TFE3 in mouse skeletal muscle through the Ca2+-stimulated protein phosphatase calcineurin. Skeletal muscle-specific ablation of TFEB exhibits impaired glucose homeostasis and mitochondrial biogenesis with reduced metabolic flexibility during exercise, and global TFE3 depletion results in diminished endurance and abolished exercise-induced metabolic benefits. Transcriptomic analysis of the muscle-specific TFEB-null mice has demonstrated that TFEB regulates the expression of genes involved in glucose metabolism and mitochondrial homeostasis. This review aims to summarize and discuss emerging roles for TFEB/TFE3 in metabolic and adaptive responses to exercise/contractile activity in skeletal muscle.
    Keywords:  AMPK; PGC1a; calcineurin; mTOR
    DOI:  https://doi.org/10.1152/ajpendo.00339.2020
  18. Antiviral Res. 2020 Aug 26. pii: S0166-3542(20)30336-3. [Epub ahead of print] 104922
    ML-SA1, a selective TRPML agonist, inhibits DENV2 and ZIKV by promoting lysosomal acidification and protease activity.
    Zhiqiang Xia, Luyao Wang, Songryong Li, Wei Tang, Fang Sun, Yingliang Wu, Lixia Miao, Zhijian Cao.
      Arboviruses, especially Dengue virus (DENV) and Zika virus (ZIKV), have been a severe threat to human health in the last few years due to uncontrollable transmission. There are no approved vaccines or clinical drugs available for use to prevent and treat their infections. Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3) were reported to modulate viral entry, but the antiviral function of these modulators was unknown. Here, we reported that ML-SA1, a TRPML agonist, inhibited DENV2 and ZIKV in vitro in a dose-dependent manner. Time-of-drug-addition experiments showed that ML-SA1 mainly restricted viral entry. Moreover, the selective TRPML3 activator SN-2 was found to share a similar antiviral effect against DENV2 and ZIKV, but the specific TRPML1 agonist MK6-83 was not effective. Although ML-SA1 was further revealed to induce autophagy, its antiviral role was independent of autophagy induction. Finally, ML-SA1 was found to inhibit DENV2 and ZIKV by promoting lysosome acidification and protease activity to cause viral degradation. Together, our study identifies two TRPML agonists, ML-SA1 and SN-2, as potent inhibitors of DENV2 and ZIKV, which may lead to the discovery of new candidates against viruses.
    Keywords:  DENV2; TRPML; ZIKV; agonist; autophagy; lysosomal acidification
    DOI:  https://doi.org/10.1016/j.antiviral.2020.104922
  19. J Nephrol. 2020 Aug 25.
    Downregulation of megalin, cubilin, ClC-5 and podocin in Fabry nephropathy: potential implications in the decreased effectiveness of enzyme replacement therapy.
    Hernán Trimarchi, Monica Ceol, Lisa Gianesello, Giovanna Priante, Alejandro Iotti, Dorella Del Prete.
      Fabry disease is an X-linked disorder due to mutations in α-galactosidase A, resulting in the accumulation of enzyme substrates and cell malfunction. Kidney involvement is frequent, affecting all native kidney cell types. Podocyte damage results in proteinuria and chronic kidney disease. End-stage kidney disease is the rule in middle-aged males and some females with the classic phenotype. In podocytes and kidney proximal tubular cells, megalin is one of the molecules involved in enzyme replacement therapy (ERT) cellular absorption. After podocyte damage, podocin concentration is decreased and contributes to progressive proteinuria. We report in a male and a female patient the decreased expression of megalin, cubilin, ClC-5 and podocin compared to controls and chronic kidney disease (CKD) biopsies. Moreover, the decrease in ClC-5, a molecule engaged in endosomal-lysosomal acidification, could also affect ERT. These findings may partially explain some of the dysfunctions described in Fabry nephropathy and could highlight possible alterations in the pharmacokinetics of the delivered enzyme.
    Keywords:  CLC-5; Chronic kidney disease; Cubilin; End-stage kidney disease; Fabry disease; Megalin; Podocin; Podocyte; Proteinuria; Synaptopodin
    DOI:  https://doi.org/10.1007/s40620-020-00835-9
  20. Cancers (Basel). 2020 Aug 24. pii: E2391. [Epub ahead of print]12(9):
    Loss of Two-Pore Channel 2 (TPC2) Expression Increases the Metastatic Traits of Melanoma Cells by a Mechanism Involving the Hippo Signalling Pathway and Store-Operated Calcium Entry.
    Antonella D'Amore, Ali Ahmed Hanbashi, Silvia Di Agostino, Fioretta Palombi, Andrea Sacconi, Aniruddha Voruganti, Marilena Taggi, Rita Canipari, Giovanni Blandino, John Parrington, Antonio Filippini.
      Melanoma is one of the most aggressive and treatment-resistant human cancers. The two-pore channel 2 (TPC2) is located on late endosomes, lysosomes and melanosomes. Here, we characterized how TPC2 knockout (KO) affected human melanoma cells derived from a metastatic site. TPC2 KO increased these cells' ability to invade the extracelullar matrix and was associated with the increased expression of mesenchymal markers ZEB-1, Vimentin and N-Cadherin, and the enhanced secretion of MMP9. TPC2 KO also activated genes regulated by YAP/TAZ, which are key regulators of tumourigenesis and metastasis. Expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-βII, part of the HIPPO pathway that negatively regulates YAP/TAZ activity, were reduced by TPC2 KO and RNA interference knockdown. We propose a cellular mechanism mediated by ORAI1/Ca2+/PKC-βII to explain these findings. Highlighting their potential clinical significance, patients with metastatic tumours showed a reduction in TPC2 expression. Our research indicates a novel role of TPC2 in melanoma. While TPC2 loss may not activate YAP/TAZ target genes in primary melanoma, in metastatic melanoma it could activate such genes and increase cancer aggressiveness. These findings aid the understanding of tumourigenesis mechanisms and could provide new diagnostic and treatment strategies for skin cancer and other metastatic cancers.
    Keywords:  HIPPO; SOCE; TPC2; melanoma; metastasis
    DOI:  https://doi.org/10.3390/cancers12092391
  21. Am J Med Genet A. 2020 Aug 28.
    Patient with an autosomal-recessive MBTPS1-linked phenotype and clinical features of Silver-Russell syndrome.
    Robert Meyer, Miriam Elbracht, Thomas Opladen, Thomas Eggermann.
      Pathogenic variants in the MBTPS1 gene encoding the Site 1 protease have been described so far only in one growth retarded patients with skeletal deformities, large ears, a triangular face reminiscent to Silver-Russell syndrome (SRS), and elevated blood lysosomal enzymes. We now report on the identification of a second adult patient homozygous for one of the two published pathogenic MBTPS1 variants (p.Asp365Gly) by Whole Exome Sequencing (WES), and a comparable phenotype. With this case, the association of pathogenic variants in MBTPS1 with a recognizable disorder could be confirmed, and the autosomal recessive inheritance is further established. As the variant was identified after a long diagnostic odyssey of the family, this example illustrates the need to apply WES in the diagnostic workup in case of growth retardation as early as possible. By compiling the clinical data of this new patient with those of the already reported patient, a better prognosis for future patients with MBTPS1 variants can be issued, and clinical management can be adjusted.
    Keywords:  MBTPS1; Silver-Russell syndrome; pathogenic variant
    DOI:  https://doi.org/10.1002/ajmg.a.61833
  22. Metabolomics. 2020 Aug 26. 16(9): 91
    Cationic amphiphilic drugs induce elevation in lysoglycerophospholipid levels and cell death in leukemia cells.
    Inger Ødum Nielsen, Line Groth-Pedersen, Jano Dicroce-Giacobini, Anna Sofie Holm Jonassen, Monika Mortensen, Mesut Bilgin, Kjeld Schmiegelow, Marja Jäättelä, Kenji Maeda.
      INTRODUCTION: Repurposing of cationic amphiphilic drugs (CADs) emerges as an attractive therapeutic solution against various cancers, including leukemia. CADs target lysosomal lipid metabolism and preferentially kill cancer cells via induction of lysosomal membrane permeabilization, but the exact effects of CADs on the lysosomal lipid metabolism remain poorly illuminated.OBJECTIVES: We aimed to systematically monitor CAD-induced alterations in the quantitative lipid profiles of leukemia cell lines in order to chart effects of CADs on the metabolism of various lipid classes present in these cells.
    METHODS: We conducted this study on eight cultured cell lines representing two leukemia types, acute lymphoblastic leukemia and acute myeloid leukemia. Mass spectrometry-based quantitative shotgun lipidomics was employed to quantify the levels of around 400 lipid species of 26 lipid classes in the leukemia cell lines treated or untreated with a CAD, siramesine.
    RESULTS: The two leukemia types displayed high, but variable sensitivities to CADs and distinct profiles of cellular lipids. Treatment with siramesine rapidly altered the levels of diverse lipid classes in both leukemia types. These included sphingolipid classes previously reported to play key roles in CAD-induced cell death, but also lipids of other categories. We demonstrated that the treatment with siramesine additionally elevated the levels of numerous cytolytic lysoglycerophospholipids in positive correlation with the sensitivity of individual leukemia cell lines to siramesine.
    CONCLUSIONS: Our study shows that CAD treatment alters balance in the metabolism of glycerophospholipids, and proposes elevation in the levels of lysoglycerophospholipids as part of the mechanism leading to CAD-induced cell death of leukemia cells.
    Keywords:  Cancer; Lipidomics; Lysoglycerophospholipids; Lysosomes; Sphingolipids; Systems biology
    DOI:  https://doi.org/10.1007/s11306-020-01710-1
  23. Autophagy. 2020 Aug 24. 1-15
    1-Deoxysphingolipids cause autophagosome and lysosome accumulation and trigger NLRP3 inflammasome activation.
    Mario A Lauterbach, Victor Saavedra, Matthew S J Mangan, Anke Penno, Christoph Thiele, Eicke Latz, Lars Kuerschner.
      1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system.ABBREVIATIONS: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N3635P: azido-STAR635P; N3Cy3: azido-cyanine 3; N3picCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.
    Keywords:  Autophagy; HSAN1; crystal; doxSA; innate immunity; lipid; macrophage
    DOI:  https://doi.org/10.1080/15548627.2020.1804677
  24. Genes (Basel). 2020 Aug 25. pii: E989. [Epub ahead of print]11(9):
    Regulation of mTORC1 by Upstream Stimuli.
    Chase H Melick, Jenna L Jewell.
      The mammalian target of rapamycin (mTOR) is an evolutionary conserved Ser/Thr protein kinase that senses multiple upstream stimuli to control cell growth, metabolism, and autophagy. mTOR is the catalytic subunit of mTOR complex 1 (mTORC1). A significant amount of research has uncovered the signaling pathways regulated by mTORC1, and the involvement of these signaling cascades in human diseases like cancer, diabetes, and ageing. Here, we review advances in mTORC1 regulation by upstream stimuli. We specifically focus on how growth factors, amino acids, G-protein coupled receptors (GPCRs), phosphorylation, and small GTPases regulate mTORC1 activity and signaling.
    Keywords:  G-protein coupled receptors; amino acids; and autophagy; cell growth; kinases; mTORC1; metabolism; phosphorylation; small GTPases
    DOI:  https://doi.org/10.3390/genes11090989
  25. Front Cell Dev Biol. 2020 ;8 715
    Mechanism of Activation of Mechanistic Target of Rapamycin Complex 1 by Methionine.
    Munehiro Kitada, Jing Xu, Yoshio Ogura, Itaru Monno, Daisuke Koya.
      Nutrients are closely involved in the regulation of lifespan and metabolic health. Cellular activities, such as the regulation of metabolism, growth, and aging, are mediated by a network of nutrients and nutrient-sensing pathways. Among the nutrient-sensing pathways, the mechanistic target of rapamycin complex 1 (mTORC1) acts as the central regulator of cellular functions, which include autophagy. Autophagy plays a significant role in the removal of protein aggregates and damaged or excess organelles, including mitochondria, to maintain intracellular homeostasis, which is involved in lifespan extension and cardiometabolic health. Moreover, dietary methionine restriction may have a beneficial effect on lifespan extension and metabolic health. In contrast, methionine may activate mTORC1 and suppress autophagy. As the mechanism of methionine sensing on mTORC1, SAMTOR was identified as a sensor of S-adenosyl methionine (SAM), a metabolite of methionine, in the cytoplasm. Conversely, methionine may activate the mTORC1 signaling pathway through the activation of phosphatase 2A (PP2A) because of increased methylation in response to intracellular SAM levels. In this review, we summarized the recent findings regarding the mechanism via which methionine activates mTORC1.
    Keywords:  S-adenosyl methionine; SAMTOR; autophagy; mechanistic target of rapamycin complex 1; methionine; phosphatase 2A methylation
    DOI:  https://doi.org/10.3389/fcell.2020.00715
  26. Transl Oncol. 2020 Aug 24. pii: S1936-5233(20)30339-9. [Epub ahead of print]13(12): 100847
    Searching for the real function of mTOR signaling in the regulation of PD-L1 expression.
    Shi-Yong Sun.
      The mammalian target of rapamycin (mTOR), via forming two important complexes: mTOR complex 1 (mTORC1) and complex 2 (mTORC2), plays an important role in the regulation of immunity in addition to exerting many other biological funcions. Beyond its regulatory effects on immune cells, the mTOR axis also regulates the expression of programmed death-ligand 1 (PD-L1) in cancer cells; accordingly, inhibition of mTOR alters PD-L1 levels in different cancer cell types. However, the currently published studies on mTOR inhibition-induced PD-L1 alteration have generated conflicting results. This review will focus on summarizing current findings in this regard and discussing possible reasons for the discrepancies and their potential implications for PD-L1 modulation in cancer therapy.
    Keywords:  Cancer; PD-L1; Rapalogs; mTOR; mTORC1; p70S6K
    DOI:  https://doi.org/10.1016/j.tranon.2020.100847
  27. Oncogene. 2020 Aug 27.
    Serum- and glucocorticoid- inducible kinase 2, SGK2, is a novel autophagy regulator and modulates platinum drugs response in cancer cells.
    Valentina Ranzuglia, Ilaria Lorenzon, Ilenia Pellarin, Maura Sonego, Alessandra Dall'Acqua, Sara D'Andrea, Sara Lovisa, Ilenia Segatto, Michela Coan, Jerry Polesel, Diego Serraino, Patrizia Sabatelli, Paola Spessotto, Barbara Belletti, Gustavo Baldassarre, Monica Schiappacassi.
      For many tumor types chemotherapy still represents the therapy of choice and many standard treatments are based on the use of platinum (PT) drugs. However, de novo or acquired resistance to platinum is frequent and leads to disease progression. In Epithelial Ovarian Cancer (EOC) patients, PT-resistant recurrences are very common and improving the response to treatment still represents an unmet clinical need. To identify new modulators of PT-sensitivity, we performed a loss-of-function screening targeting 680 genes potentially involved in the response of EOC cells to platinum. We found that SGK2 (Serum-and Glucocorticoid-inducible kinase 2) plays a key role in PT-response. We show here that EOC cells relay on the induction of autophagy to escape PT-induced death and that SGK2 inhibition increases PT sensitivity inducing a block in the autophagy cascade due to the impairment of lysosomal acidification. Mechanistically we demonstrate that SGK2 controls autophagy in a kinase-dependent manner by binding and inhibiting the V-ATPase proton pump. Accordingly, SGK2 phosphorylates the subunit V1H (ATP6V1H) of V-ATPase and silencing or chemical inhibition of SGK2, affects the normal autophagic flux and sensitizes EOC cells to platinum. Hence, we identified a new pathway that links autophagy to the survival of cancer cells under platinum treatment in which the druggable kinase SGK2 plays a central role. Our data suggest that blocking autophagy via SGK2 inhibition could represent a novel therapeutic strategy to improve patients' response to platinum.
    DOI:  https://doi.org/10.1038/s41388-020-01433-6
  28. Int Rev Neurobiol. 2020 ;pii: S0074-7742(20)30009-X. [Epub ahead of print]155 1-35
    Dysregulation of metabolic flexibility: The impact of mTOR on autophagy in neurodegenerative disease.
    Kenneth Maiese.
      Non-communicable diseases (NCDs) that involve neurodegenerative disorders and metabolic disease impact over 400 million individuals globally. Interestingly, metabolic disorders, such as diabetes mellitus, are significant risk factors for the development of neurodegenerative diseases. Given that current therapies for these NCDs address symptomatic care, new avenues of discovery are required to offer treatments that affect disease progression. Innovative strategies that fill this void involve the mechanistic target of rapamycin (mTOR) and its associated pathways of mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2), AMP activated protein kinase (AMPK), trophic factors that include erythropoietin (EPO), and the programmed cell death pathways of autophagy and apoptosis. These pathways are intriguing in their potential to provide effective care for metabolic and neurodegenerative disorders. Yet, future work is necessary to fully comprehend the entire breadth of the mTOR pathways that can effectively and safely translate treatments to clinical medicine without the development of unexpected clinical disabilities.
    Keywords:  AMPK; Alzheimer's disease; Apoptosis; Autophagy; Dementia; Diabetes mellitus; Erythropoietin; mTOR; mTORC1; mTORC2
    DOI:  https://doi.org/10.1016/bs.irn.2020.01.009
  29. Cell Signal. 2020 Aug 19. pii: S0898-6568(20)30221-7. [Epub ahead of print] 109744
    HGF protected against diabetic nephropathy via autophagy-lysosome pathway in podocyte by modulating PI3K/Akt-GSK3β-TFEB axis.
    Bo Hou, Yankun Li, Xue Li, Congying Zhang, Zhonghua Zhao, Qi Chen, Nong Zhang, Hui Li.
      Podocyte loss is a detrimental feature and major cause of proteinuria in diabetic nephropathy (DN). Our previous study revealed that hepatocyte growth factor (HGF) prevented high glucose-induced podocyte injury via enhancing autophagy. In the current study, we aimed to assess the role of HGF on podocyte homeostasis in DN and clarify its mechanisms further. Diabetic mice treated with HGF had markedly reduced ratio of kidney weight to body weight, urinary albumin excretion, podocyte loss and matrix expansion compared with that in the non-treated counterpart. Simultaneously, HGF-treated diabetic mice exhibited increased autophagy activity as indicated by the decreased accumulation of sequestosome 1 (SQSTM1/ p62) and increased microtubule-associated proteins 1 light chains 3 (LC3) II/LC3I ratio. These beneficial effects of HGF were blocked by HGF/c-Met inhibitor Crizotinib or phosphatidylinositide 3-kinases (PI3K) inhibitor LY294002. Moreover, HGF treatment obviously prevented inactivation of the protein kinase B (Akt)-glycogen synthase kinase 3 beta (GSK3β)-transcription factor EB (TFEB) axis in high glucose-stimulated podocytes, which was associated with improved lysosome function and autophagy. Accordingly, adenovirus vector encoding constitutively active GSK3β (Ad-GSK3β-S9A) offset whereas small interfering RNA against GSK3β (GSK3β siRNA) recapitulated salutary effects of HGF on lysosome number and autophagy in podocytes. These results suggested that HGF protected against diabetic nephropathy through restoring podocyte autophagy, which at least partially involved PI3K/Akt-GSK3β-TFEB axis-mediated lysosomal function improvement.
    Keywords:  Autophagy; Diabetic nephropathy; Glycogen synthase kinase 3 beta; Hepatocyte growth factor; Lysosome; Podocyte
    DOI:  https://doi.org/10.1016/j.cellsig.2020.109744
  30. Biochem Biophys Res Commun. 2020 Aug 25. pii: S0006-291X(20)31261-4. [Epub ahead of print]
    GOLPH3 silencing inhibits adhesion of glioma U251 cells by regulating ITGB1 degradation under serum starvation.
    Wenjian Zhan, Jilun Zhang, Yongchuan Luo, Rutong Yu.
      GOLPH3, an oncoprotein, plays crucial roles in tumor etiology. Compelling evidences have demonstrated that GOLPH3 contributes to regulate tumor cell growth, migration and invasion under normal nutrient condition. However, the oncogenic activity of GOLPH3 under serum starvation remains largely unknown. In this study, we reported that GOLPH3 depletion led to marked reduction in adhesion of glioma U251 cells, particularly under serum deprivation. We found that silencing of GOLPH3 expression reduced the protein amount of ITGB1 only in serum-free medium. Further insights into the mechanism between GOLPH3 and ITGB1, we applied proteasome or lysosome inhibitor to block the degradation of ITGB1, and identified GOLPH3 silencing can prompt ITGB1 lysosomal degradation under serum starvation. Finally, we found the reductions in glioma cell adhesion and ITGB1 protein amount could be rescued by ITGB1 overexpression. Taken together, these results show that GOLPH3 contributes to the adhesion of glioma cells by regulating the lysosomal degradation of ITGB1 under serum starvation.
    Keywords:  Adhesion; GOLPH3; Glioma; ITGB1; Lysosomal degaradation
    DOI:  https://doi.org/10.1016/j.bbrc.2020.06.044
  31. Front Aging Neurosci. 2020 ;12 230
    Rapamycin, by Inhibiting mTORC1 Signaling, Prevents the Loss of Striatal Bidirectional Synaptic Plasticity in a Rat Model of L-DOPA-Induced Dyskinesia.
    Valeria Calabrese, Anna Di Maio, Gioia Marino, Antonella Cardinale, Giuseppina Natale, Arianna De Rosa, Federica Campanelli, Maria Mancini, Francesco Napolitano, Luigi Avallone, Paolo Calabresi, Alessandro Usiello, Veronica Ghiglieri, Barbara Picconi.
      Levodopa (L-DOPA) treatment is the main gold-standard therapy for Parkinson disease (PD). Besides good antiparkinsonian effects, prolonged use of this drug is associated to the development of involuntary movements known as L-DOPA-induced dyskinesia (LID). L-DOPA-induced dyskinesia is linked to a sensitization of dopamine (DA) D1 receptors located on spiny projection neurons (SPNs) of the dorsal striatum. Several evidences have shown that the emergence of LID can be related to striatal D1/cAMP/PKA/DARPP-32 and extracellular signal-regulated kinases (ERK1/2) pathway overactivation associated to aberrant N-methyl-d-aspartate (NMDA) receptor function. In addition, within striatum, ERK1/2 is also able to modulate in a D1 receptor-dependent manner the activity of the mammalian target of rapamycin complex 1 (mTORC1) pathway under DA depletion and L-DOPA therapy. Consistently, increased mTORC1 signaling appears during chronic administration of L-DOPA and shows a high correlation with the severity of dyskinesia. Furthermore, the abnormal activation of the D1/PKA/DARPP-32 cascade is paralleled by increased phosphorylation of the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor at the PKA Ser845 site. The GluA1 promotes excitatory AMPA receptor-mediated transmission and may be implicated in the alterations found at the corticostriatal synapses of dyskinetic animals. In our study, we investigated the role of mTORC1 pathway activation in modulating bidirectional striatal synaptic plasticity in L-DOPA-treated parkinsonian rats. Inhibition of mTORC1 by coadministration of rapamycin to L-DOPA was able to limit the magnitude of LID expression, accounting for a therapeutic effect of this drug. In particular, behavioral data showed that, in L-DOPA-treated rats, rapamycin administration induced a selective decrease of distinct components of abnormal involuntary movements (i.e., axial and orolingual dyskinesia). Furthermore, ex vivo patch clamp and intracellular recordings of SPNs revealed that pharmacological inhibition of mTORC1 also resulted associated with a physiological bidirectional plasticity, when compared to dyskinetic rats treated with L-DOPA alone. This study uncovers the important role of mTORC1 inhibition to prevent the loss of striatal bidirectional plasticity under chronic L-DOPA treatment in rodent models of PD.
    Keywords:  Parkinson’s disease; depotentiation; dorsolateral striatum; functional plasticity; levodopa-induced dyskinesia
    DOI:  https://doi.org/10.3389/fnagi.2020.00230
  32. Hum Cell. 2020 Aug 26.
    Prevention of Akt phosphorylation is a key to targeting cancer stem-like cells by mTOR inhibition.
    Shyuichiro Matsubara, Koichiro Tsukasa, Taisaku Kuwahata, Sonshin Takao.
      CD133 expression in pancreatic cancer correlates with poor prognosis and increased metastasis. CD133+ pancreatic cancer cells exhibit cancer stem cell (CSC)-like properties. We established a CD133+ cell-rich subline from Capan-1 pancreatic cancer cells as a pancreatic CSC model and compared the effects of KU-0063794, a dual mTORC1/mTORC2 inhibitor, against those of mTORC1-specific rapamycin. We found that KU-0063794 prevents sphere formation, a self-renewal index, at high concentrations. Rapamycin inhibited sphere formation but to a lesser degree. In the present study, we aimed to determine the mechanistic roles of mTOR complex 2 (mTORC2) in maintaining CSC-like properties. By examining the PI3K/Akt/mTOR signaling pathway, we observed lower Akt phosphorylation in KU-0063794-treated cells. Phosphorylation of mTORC1 downstream effectors was inhibited by both inhibitors. Thus, mTORC2 activates Akt and modulate stem-like properties, whereas mTORC1 downstream signaling correlates directly with stem-like properties.
    Keywords:  CD133; Mechanistic/mammalian target of rapamycin (mTOR); Pancreatic cancer stem cells; mTOR complex 1 (mTORC1); mTOR complex 2 (mTORC2)
    DOI:  https://doi.org/10.1007/s13577-020-00416-9
  33. J Biol Chem. 2020 Aug 28. 295(35): 12353-12354
    To cut or not to cut: New rules for proteolytic shedding of membrane proteins.
    Stefan F Lichtenthaler, Edgar Meinl.
      Sheddases are specialized proteases that control the abundance and function of membrane proteins by cleaving their substrate's extracellular domain (ectodomain), a process known as shedding. Hundreds of shedding substrates have been identified, but little is known about the mechanisms that govern ectodomain shedding. Iwagishi et al. now report that negatively charged amino acids in the membrane-proximal juxtamembrane domain of substrates make them resistant to shedding by the metalloprotease ADAM17. These findings will help researchers better understand the regulation of shedding and may aid in the development of drugs targeting sheddases.
    DOI:  https://doi.org/10.1074/jbc.H120.015304
  34. Nat Commun. 2020 Aug 28. 11(1): 4332
    Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection.
    Piero Giansanti, Jeroen R P M Strating, Kyra A Y Defourny, Ieva Cesonyte, Alexia M S Bottino, Harm Post, Ekaterina G Viktorova, Vien Quang Tri Ho, Martijn A Langereis, George A Belov, Esther N M Nolte-'t Hoen, Albert J R Heck, Frank J M van Kuppeveld.
      The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.
    DOI:  https://doi.org/10.1038/s41467-020-18168-3
  35. Cell Rep. 2020 Aug 25. pii: S2211-1247(20)31044-5. [Epub ahead of print]32(8): 108059
    Mitochondrial Regulation of the 26S Proteasome.
    Thomas Meul, Korbinian Berschneider, Sabine Schmitt, Christoph H Mayr, Laura F Mattner, Herbert B Schiller, Ayse S Yazgili, Xinyuan Wang, Christina Lukas, Camille Schlesser, Cornelia Prehn, Jerzy Adamski, Elisabeth Graf, Thomas Schwarzmayr, Fabiana Perocchi, Alexandra Kukat, Aleksandra Trifunovic, Laura Kremer, Holger Prokisch, Bastian Popper, Christine von Toerne, Stefanie M Hauck, Hans Zischka, Silke Meiners.
      The proteasome is the main proteolytic system for targeted protein degradation in the cell and is fine-tuned according to cellular needs. Here, we demonstrate that mitochondrial dysfunction and concomitant metabolic reprogramming of the tricarboxylic acid (TCA) cycle reduce the assembly and activity of the 26S proteasome. Both mitochondrial mutations in respiratory complex I and treatment with the anti-diabetic drug metformin impair 26S proteasome activity. Defective 26S assembly is reversible and can be overcome by supplementation of aspartate or pyruvate. This metabolic regulation of 26S activity involves specific regulation of proteasome assembly factors via the mTORC1 pathway. Of note, reducing 26S activity by metformin confers increased resistance toward the proteasome inhibitor bortezomib, which is reversible upon pyruvate supplementation. Our study uncovers unexpected consequences of defective mitochondrial metabolism for proteasomal protein degradation in the cell, which has important pathophysiological and therapeutic implications.
    Keywords:  26S proteasome; Rpn6; TCA; aspartate; metabolic reprogramming; metformin; mitochondria; proteasome assembly factors; proteasome inhibitor resistance; pyruvate; respiratory complex I
    DOI:  https://doi.org/10.1016/j.celrep.2020.108059
  36. J Psychopharmacol. 2020 Aug 27. 269881120944159
    The role of the mTOR pathway in models of drug-induced reward and the behavioural constituents of addiction.
    Marcos Ucha, David Roura-Martínez, Emilio Ambrosio, Alejandro Higuera-Matas.
      BACKGROUND: Exposure to drugs of abuse induces neuroadaptations in critical nodes of the so-called reward systems that are thought to mediate the transition from controlled drug use to the compulsive drug-seeking that characterizes addictive disorders. These neural adaptations are likely to require protein synthesis, which is regulated, among others, by the mechanistic target of the rapamycin kinase (mTOR) signalling cascade.METHODS: We have performed a narrative review of the literature available in PubMed about the involvement of the mTOR pathway in drug-reward and addiction-related phenomena.
    AIMS: The aim of this study was to review the underlying architecture of this complex intracellular network and to discuss the alterations of its components that are evident after exposure to drugs of abuse. The aim was also to delineate the effects that manipulations of the mTOR network have on models of drug reward and on paradigms that recapitulate some of the psychological components of addiction.
    RESULTS: There is evidence for the involvement of the mTOR pathway in the acute and rewarding effects of drugs of abuse, especially psychostimulants. However, the data regarding opiates are scarce. There is a need to use sophisticated animal models of addiction to ascertain the real role of the mTOR pathway in this pathology and not just in drug-mediated reward. The involvement of this pathway in behavioural addictions and impulsivity should also be studied in detail in the future.
    CONCLUSIONS: Although there is a plethora of data about the modulation of mTOR by drugs of abuse, the involvement of this signalling pathway in addictive disorders requires further research.
    Keywords:  addiction; drugs of abuse; mTOR; mTORC1; mTORC2; protein synthesis; rapamycin
    DOI:  https://doi.org/10.1177/0269881120944159
  37. Neuron. 2020 Aug 19. pii: S0896-6273(20)30567-5. [Epub ahead of print]
    Expression of the Neuronal tRNA n-Tr20 Regulates Synaptic Transmission and Seizure Susceptibility.
    Mridu Kapur, Archan Ganguly, Gabor Nagy, Scott I Adamson, Jeffrey H Chuang, Wayne N Frankel, Susan L Ackerman.
      The mammalian genome has hundreds of nuclear-encoded tRNAs, but the contribution of individual tRNA genes to cellular and organismal function remains unknown. Here, we demonstrate that mutations in a neuronally enriched arginine tRNA, n-Tr20, increased seizure threshold and altered synaptic transmission. n-Tr20 expression also modulated seizures caused by an epilepsy-linked mutation in Gabrg2, a gene encoding a GABAA receptor subunit. Loss of n-Tr20 altered translation initiation by activating the integrated stress response and suppressing mTOR signaling, the latter of which may contribute to altered neurotransmission in mutant mice. Deletion of a highly expressed isoleucine tRNA similarly altered these signaling pathways in the brain, suggesting that regulation of translation initiation is a conserved response to tRNA loss. Our data indicate that loss of a single member of a tRNA family results in multiple cellular phenotypes, highlighting the disease-causing potential of tRNA mutations.
    Keywords:  C57BL/6J; C57BL/6N; Chromosome 1; GCN2; electroconvulsive seizure threshold; excitatory inhibitory balance; quantitative trait loci; ribosome stalling; tRNA isodecoder; translation elongation
    DOI:  https://doi.org/10.1016/j.neuron.2020.07.023
  38. Cell Stem Cell. 2020 Aug 19. pii: S1934-5909(20)30398-2. [Epub ahead of print]
    Defects in mRNA Translation in LRRK2-Mutant hiPSC-Derived Dopaminergic Neurons Lead to Dysregulated Calcium Homeostasis.
    Jungwoo Wren Kim, Xiling Yin, Aanishaa Jhaldiyal, Mohammed Repon Khan, Ian Martin, Zhong Xie, Tamara Perez-Rosello, Manoj Kumar, Leire Abalde-Atristain, Jinchong Xu, Li Chen, Stephen M Eacker, D James Surmeier, Nicholas T Ingolia, Ted M Dawson, Valina L Dawson.
      The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). This mutation results in dopaminergic neurodegeneration via dysregulated protein translation, although how alterations in protein synthesis contribute to neurodegeneration in human neurons is not known. Here we define the translational landscape in LRRK2-mutant dopaminergic neurons derived from human induced pluripotent stem cells (hiPSCs) via ribosome profiling. We found that mRNAs that have complex secondary structure in the 5' untranslated region (UTR) are translated more efficiently in G2019S LRRK2 neurons. This leads to the enhanced translation of multiple genes involved in Ca2+ regulation and to increased Ca2+ influx and elevated intracellular Ca2+ levels, a major contributor to PD pathogenesis. This study reveals a link between dysregulated translation control and Ca2+ homeostasis in G2019S LRRK2 human dopamine neurons, which potentially contributes to the progressive and selective dopaminergic neurotoxicity in PD.
    Keywords:  5′ UTR; LRRK2; Parkinson’s disease; RPS15; calcium homeostasis; ribosome profiling; translatome; uS19
    DOI:  https://doi.org/10.1016/j.stem.2020.08.002
  39. Front Cell Dev Biol. 2020 ;8 779
    The RUFYs, a Family of Effector Proteins Involved in Intracellular Trafficking and Cytoskeleton Dynamics.
    Rémy Char, Philippe Pierre.
      Intracellular trafficking is essential for cell structure and function. In order to perform key tasks such as phagocytosis, secretion or migration, cells must coordinate their intracellular trafficking, and cytoskeleton dynamics. This relies on certain classes of proteins endowed with specialized and conserved domains that bridge membranes with effector proteins. Of particular interest are proteins capable of interacting with membrane subdomains enriched in specific phosphatidylinositol lipids, tightly regulated by various kinases and phosphatases. Here, we focus on the poorly studied RUFY family of adaptor proteins, characterized by a RUN domain, which interacts with small GTP-binding proteins, and a FYVE domain, involved in the recognition of phosphatidylinositol 3-phosphate. We report recent findings on this protein family that regulates endosomal trafficking, cell migration and upon dysfunction, can lead to severe pathology at the organismal level.
    Keywords:  FYVE; RUFY; RUN; cancer; cytoskeleton; immunity; neurodegenerative diseases; phosphatidylinositol 3-phosphate
    DOI:  https://doi.org/10.3389/fcell.2020.00779
  40. Nature. 2020 Aug 26.
    Structure of the C9orf72 ARF GAP complex that is haploinsufficient in ALS and FTD.
    Ming-Yuan Su, Simon A Fromm, Roberto Zoncu, James H Hurley.
      Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a β-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.
    DOI:  https://doi.org/10.1038/s41586-020-2633-x
  41. Autophagy. 2020 Aug 25. 1-3
    Structure of human ATG9A: how holey art thou?
    Wayne D Hawkins, Daniel J Klionsky.
      Several studies have provided insight into the unique intracellular localization, dynamic trafficking and diverse repertoire of binding partners of Atg9/ATG9, but structural details of the protein have remained elusive. Guardia and colleagues now report the structure of human ATG9A to a resolution of 2.9 Å, revealing, among other features, an elaborate system of tunnels permeating the ATG9A protein complex.
    Keywords:  Autophagy; lipid transfer; lysosome; membrane protein; protein structure; stress
    DOI:  https://doi.org/10.1080/15548627.2020.1810901
  42. BMC Biol. 2020 Aug 28. 18(1): 107
    Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy.
    Dan Li, Shu-Gao Yang, Cheng-Wen He, Zheng-Tan Zhang, Yongheng Liang, Hui Li, Jing Zhu, Xiong Su, Qingqiu Gong, Zhiping Xie.
      BACKGROUND: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive.RESULTS: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response.
    CONCLUSIONS: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.
    Keywords:  Autophagy; Glycerolipid; Intracellular trafficking; Organelle; Phospholipid
    DOI:  https://doi.org/10.1186/s12915-020-00837-w
  43. Front Cell Dev Biol. 2020 ;8 757
    Membrane Heterogeneity Controls Cellular Endocytic Trafficking.
    Gregory M I Redpath, Verena M Betzler, Pascal Rossatti, Jérémie Rossy.
      Endocytic trafficking relies on highly localized events in cell membranes. Endocytosis involves the gathering of protein (cargo/receptor) at distinct plasma membrane locations defined by specific lipid and protein compositions. Simultaneously, the molecular machinery that drives invagination and eventually scission of the endocytic vesicle assembles at the very same place on the inner leaflet of the membrane. It is membrane heterogeneity - the existence of specific lipid and protein domains in localized regions of membranes - that creates the distinct molecular identity required for an endocytic event to occur precisely when and where it is required rather than at some random location within the plasma membrane. Accumulating evidence leads us to believe that the trafficking fate of internalized proteins is sealed following endocytosis, as this distinct membrane identity is preserved through the endocytic pathway, upon fusion of endocytic vesicles with early and sorting endosomes. In fact, just like at the plasma membrane, multiple domains coexist at the surface of these endosomes, regulating local membrane tubulation, fission and sorting to recycling pathways or to the trans-Golgi network via late endosomes. From here, membrane heterogeneity ensures that fusion events between intracellular vesicles and larger compartments are spatially regulated to promote the transport of cargoes to their intracellular destination.
    Keywords:  CLIC/GEEC; Rab11; clathrin; endophilin; endosomal sorting; phosphatidylserine; phosphoinositide; retromer/retriever
    DOI:  https://doi.org/10.3389/fcell.2020.00757
  44. Int J Mol Sci. 2020 Aug 25. pii: E6113. [Epub ahead of print]21(17):
    Lipophagy and Lipolysis Status in Lipid Storage and Lipid Metabolism Diseases.
    Anna Kloska, Magdalena Węsierska, Marcelina Malinowska, Magdalena Gabig-Cimińska, Joanna Jakóbkiewicz-Banecka.
      This review discusses how lipophagy and cytosolic lipolysis degrade cellular lipids, as well as how these pathway ys communicate, how they affect lipid metabolism and energy homeostasis in cells and how their dysfunction affects the pathogenesis of lipid storage and lipid metabolism diseases. Answers to these questions will likely uncover novel strategies for the treatment of aforementioned human diseases, but, above all, will avoid destructive effects of high concentrations of lipids-referred to as lipotoxicity-resulting in cellular dysfunction and cell death.
    Keywords:  TFEB; lipid droplets; lipid metabolism; lipid metabolism diseases; lipid storage diseases; lipolysis; lipophagy; mTORC1
    DOI:  https://doi.org/10.3390/ijms21176113
  45. J Cell Biol. 2020 Sep 07. pii: e202008031. [Epub ahead of print]219(9):
    Mitophagy is induced by short ubiquitin chains on mitochondria.
    Fumiyo Ikeda.
      Mitophagy has a critical role in maintaining cellular homeostasis by removing damaged mitochondria. In this issue, Yamano et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201912144) uncover that a novel complex of the autophagy adaptor optineurin and the membrane protein ATG9A specifically regulate ubiquitin-induced mitophagy.
    DOI:  https://doi.org/10.1083/jcb.202008031
  46. Curr Top Med Chem. 2020 Aug 25.
    An insight of scientific developments in TSC for better therapeutic strategy.
    Nalini Natarajan, Vijay Thiruvenkatam.
      Tuberous sclerosis complex (TSC) is a rare genetic disease, which is characterized by non-cancerous tumors in multi-organ systems in the body. Mutations in the TSC1 or TSC2 genes are known to cause the disease. The resultant mutant proteins TSC1 (hamartin) and TSC2 (tuberin) complex evades its normal tumor suppressor function, which leads to abnormal cell growth and proliferation. Both TSC1 and TSC2 are involved in several protein-protein interactions, which play a significant role in maintaining the cellular homeostasis. The recent biochemical, genetic, structural biology, clinical and drug discovery advancements on TSC give a useful insight of the disease as well as the molecular aspects of TSC1 and TSC2. The complex nature of TSC disease, wide range of manifestations, mosaicism and several other factors limit the treatment choices. This review is a compilation of the course of TSC starting from its discovery to the current findings that would take us a step ahead in finding a cure for TSC.
    Keywords:  Tuberous sclerosis complex; coiled-coil sequence; crystal structure; disruptive mutation; heat shock proteins.; hypoxia; mTOR inhibition; protein-protein interaction; rapalogs treatment
    DOI:  https://doi.org/10.2174/1568026620666200825170355
  47. Cells. 2020 Aug 21. pii: E1940. [Epub ahead of print]9(9):
    AMPK-Dependent Mechanisms but Not Hypothalamic Lipid Signaling Mediates GH-Secretory Responses to GHRH and Ghrelin.
    María J Vázquez, Marta G Novelle, Francisca Rodríguez-Pacheco, Ricardo Lage, Luis Varela, Miguel López, Leonor Pinilla, Manuel Tena-Sempere, Carlos Diéguez.
      GH (growth hormone) secretion/action is modulated by alterations in energy homeostasis, such as malnutrition and obesity. Recent data have uncovered the mechanism by which hypothalamic neurons sense nutrient bioavailability, with a relevant contribution of AMPK (AMP-activated protein kinase) and mTOR (mammalian Target of Rapamycin), as sensors of cellular energy status. However, whether central AMPK-mediated lipid signaling and mTOR participate in the regulation of pituitary GH secretion remains unexplored. We provide herein evidence for the involvement of hypothalamic AMPK signaling, but not hypothalamic lipid metabolism or CPT-1 (carnitine palmitoyltransferase I) activity, in the regulation of GH stimulatory responses to the two major elicitors of GH release in vivo, namely GHRH (growth hormone-releasing hormone) and ghrelin. This effect appeared to be GH-specific, as blocking of hypothalamic AMPK failed to influence GnRH (gonadotropin-releasing hormone)-induced LH (luteinizing hormone) secretion. Additionally, central mTOR inactivation did not alter GH responses to GHRH or ghrelin, nor this blockade affected LH responses to GnRH in vivo. In sum, we document here for the first time the indispensable and specific role of preserved central AMPK, but not mTOR, signaling, through a non-canonical lipid signaling pathway, for proper GH responses to GHRH and ghrelin in vivo.
    Keywords:  AMPK; GH; GHRH; ghrelin; hypothalamic signaling
    DOI:  https://doi.org/10.3390/cells9091940
  48. J Immunol. 2020 Aug 24. pii: ji1901480. [Epub ahead of print]
    Regulatory T Cell Stability and Migration Are Dependent on mTOR.
    Romain Vallion, Jordane Divoux, Salomé Glauzy, Emilie Ronin, Yannis Lombardi, Martina Lubrano di Ricco, Sylvie Grégoire, Ivan Nemazanyy, Aurélie Durand, Delphine Fradin, Bruno Lucas, Benoit L Salomon.
      CD4+ Foxp3+ regulatory T cells (Treg) are essential to maintain immune tolerance, as their loss leads to a fatal autoimmune syndrome in mice and humans. Conflicting findings have been reported concerning their metabolism. Some reports found that Treg have low mechanistic target of rapamycin (mTOR) activity and would be less dependent on this kinase compared with conventional T cells, whereas other reports suggest quite the opposite. In this study, we revisited this question by using mice that have a specific deletion of mTOR in Treg. These mice spontaneously develop a severe and systemic inflammation. We show that mTOR expression by Treg is critical for their differentiation into effector Treg and their migration into nonlymphoid tissues. We also reveal that mTOR-deficient Treg have reduced stability. This loss of Foxp3 expression is associated with partial Foxp3 DNA remethylation, which may be due to an increased activity of the glutaminolysis pathway. Thus, our work shows that mTOR is crucial for Treg differentiation, migration, and identity and that drugs targeting this metabolism pathway will impact on their biology.
    DOI:  https://doi.org/10.4049/jimmunol.1901480
  49. Int J Mol Sci. 2020 Aug 25. pii: E6130. [Epub ahead of print]21(17):
    Sestrin2 Phosphorylation by ULK1 Induces Autophagic Degradation of Mitochondria Damaged by Copper-Induced Oxidative Stress.
    Heejeong Kim, Byeong Tak Jeon, Isaac M Kim, Sydney J Bennett, Carolyn M Lorch, Martonio Ponte Viana, Jacob F Myers, Caroline J Trupp, Zachary T Whipps, Mondira Kundu, Soonkyu Chung, Xinghui Sun, Oleh Khalimonchuk, Jaekwon Lee, Seung-Hyun Ro.
      Selective autolysosomal degradation of damaged mitochondria, also called mitophagy, is an indispensable process for maintaining integrity and homeostasis of mitochondria. One well-established mechanism mediating selective removal of mitochondria under relatively mild mitochondria-depolarizing stress is PINK1-Parkin-mediated or ubiquitin-dependent mitophagy. However, additional mechanisms such as LC3-mediated or ubiquitin-independent mitophagy induction by heavy environmental stress exist and remain poorly understood. The present study unravels a novel role of stress-inducible protein Sestrin2 in degradation of mitochondria damaged by transition metal stress. By utilizing proteomic methods and studies in cell culture and rodent models, we identify autophagy kinase ULK1-mediated phosphorylation sites of Sestrin2 and demonstrate Sestrin2 association with mitochondria adaptor proteins in HEK293 cells. We show that Ser-73 and Ser-254 residues of Sestrin2 are phosphorylated by ULK1, and a pool of Sestrin2 is strongly associated with mitochondrial ATP5A in response to Cu-induced oxidative stress. Subsequently, this interaction promotes association with LC3-coated autolysosomes to induce degradation of mitochondria damaged by Cu-induced ROS. Treatment of cells with antioxidants or a Cu chelator significantly reduces Sestrin2 association with mitochondria. These results highlight the ULK1-Sestrin2 pathway as a novel stress-sensing mechanism that can rapidly induce autophagic degradation of mitochondria under severe heavy metal stress.
    Keywords:  ATP5A; Sestrin2; ULK1; autophagy; mitochondria; phosphorylation
    DOI:  https://doi.org/10.3390/ijms21176130
  50. Biochim Biophys Acta Mol Basis Dis. 2020 Aug 22. pii: S0925-4439(20)30300-8. [Epub ahead of print] 165952
    Identification of Annexin A2 as a key mTOR target to induce autophagy to autophagy dependent cell death in stress.
    Subhadip Mukhopadhyay, Prakash P Praharaj, Prajna P Naik, Sarmistha Talukdar, Luni Emdad, Swadesh K Das, Paul B Fisher, Sujit K Bhutia.
      Autophagy can either be cytoprotective or promote cell death in a context-dependent manner in response to stress. How autophagy leads to autophagy dependent cell death requires further clarification. In this study, we document a nonlinear roller coaster form of autophagy oscillation when cells are subjected to different stress conditions. Serum starvation induces an initial primary autophagic peak at 6 h, that helps to replenish cells with de novo fluxed nutrients, but protracted stress lead to a secondary autophagic peak around 48 h. Time kinetic studies indicate that the primary autophagic peak is reversible, whereas the secondary autophagic peak is irreversible and leads to cell death. Key players involved in different stages of autophagy including initiation, elongation and degradation during this oscillatory sequence were identified. A similar molecular pattern was intensified under apoptosis-deficient conditions. mTOR was the central molecule regulating this autophagic activity, and upon knockdown a steady increase of autophagy without any non-linear fluctuation was evident. An unbiased proteome screening approach was employed to identify the autophagy molecules potentially regulating these autophagic peaks. Our proteomics analysis has identified Annexin A2 as a stress-induced protein and its deficiency reduces autophagy and autophagy dependent cell death. Moreover, we report that mTOR in its phosphorylated condition interacts with Annexin A2 to induce autophagic fluctuation by altering its cellular localization. The work highlights the molecular mechanism of a mTOR-dependent roller coaster fluctuation of autophagy and autophagy dependent cell death during prolong stress.
    Keywords:  Annexin A2; Autophagy; Autophagy dependent cell death; Serum starvation; Stress; mTOR
    DOI:  https://doi.org/10.1016/j.bbadis.2020.165952
  51. Cell Signal. 2020 Aug 25. pii: S0898-6568(20)30223-0. [Epub ahead of print] 109746
    Dysregulation of translational control signaling in autism spectrum disorders.
    Mehdi Hooshmandi, Calvin Wong, Arkady Khoutorsky.
      Deviations from the optimal level of mRNA translation are linked to disorders with high rates of autism. Loss of function mutations in genes encoding translational repressors such as PTEN, TSC1, TSC2, and FMRP are associated with autism spectrum disorders (ASDs) in humans and their deletion in animals recapitulates many ASD-like phenotypes. Importantly, the activity of key translational control signaling pathways such as PI3K-mTORC1 and ERK is frequently dysregulated in autistic patients and animal models and their normalization rescues many abnormal phenotypes, suggesting a causal relationship. Mutations in several genes encoding proteins not directly involved in translational control have also been shown to mediate ASD phenotypes via altered signaling upstream of translation. This raises the possibility that the dysregulation of translational control signaling is a converging mechanism not only in familiar but also in sporadic forms of autism. Here, we overview the current knowledge on translational signaling in ASD and highlight how correcting the activity of key pathways upstream of translation reverses distinct ASD-like phenotypes.
    DOI:  https://doi.org/10.1016/j.cellsig.2020.109746
  52. Front Cell Dev Biol. 2020 ;8 776
    Comprehensive Model for Epidermal Growth Factor Receptor Ligand Binding Involving Conformational States of the Extracellular and the Kinase Domains.
    Tímea Hajdu, Tímea Váradi, István Rebenku, Tamás Kovács, János Szöllösi, Peter Nagy.
      The epidermal growth factor (EGF) receptor (EGFR) undergoes ligand-dependent dimerization to initiate transmembrane signaling. Although crystallographic structures of the extracellular and kinase domains are available, ligand binding has not been quantitatively analyzed taking the influence of both domains into account. Here, we developed a model explicitly accounting for conformational changes of the kinase and extracellular domains, their dimerizations and ligand binding to monomeric and dimeric receptor species. The model was fitted to ligand binding data of suspended cells expressing receptors with active or inactive kinase conformations. Receptor dimers with inactive, symmetric configuration of the kinase domains exhibit positive cooperativity and very weak binding affinity for the first ligand, whereas dimers with active, asymmetric kinase dimers are characterized by negative cooperativity and subnanomolar binding affinity for the first ligand. The homodimerization propensity of EGFR monomers with active kinase domains is ∼100-times higher than that of dimers with inactive kinase domains. Despite this fact, constitutive, ligand-independent dimers are mainly generated from monomers with inactive kinase domains due to the excess of such monomers in the membrane. The experimental finding of increased positive cooperativity at high expression levels of EGFR was recapitulated by the model. Quantitative prediction of ligand binding to different receptor species revealed that EGF binds to receptor monomers and dimers in an expression-level dependent manner without significant recruitment of monomers to dimers upon EGF stimulation below the phase transition temperature of the membrane. Results of the fitting offer unique insight into the workings of the EGFR.
    Keywords:  EGF receptor; cooperativity; dimerization; kinase domain; ligand binding
    DOI:  https://doi.org/10.3389/fcell.2020.00776
  53. Nutrients. 2020 Aug 26. pii: E2597. [Epub ahead of print]12(9):
    Docosahexaenoic Acid, a Potential Treatment for Sarcopenia, Modulates the Ubiquitin-Proteasome and the Autophagy-Lysosome Systems.
    Jung Hoon Lee, Jun Hyoung Jeon, Min Jae Lee.
      One of the characteristic features of aging is the progressive loss of muscle mass, a nosological syndrome called sarcopenia. It is also a pathologic risk factor for many clinically adverse outcomes in older adults. Therefore, delaying the loss of muscle mass, through either boosting muscle protein synthesis or slowing down muscle protein degradation using nutritional supplements could be a compelling strategy to address the needs of the world's aging population. Here, we review the recently identified properties of docosahexaenoic acid (DHA). It was shown to delay muscle wasting by stimulating intermediate oxidative stress and inhibiting proteasomal degradation of muscle proteins. Both the ubiquitin-proteasome and the autophagy-lysosome systems are modulated by DHA. Collectively, growing evidence indicates that DHA is a potent pharmacological agent that could improve muscle homeostasis. Better understanding of cellular proteolytic systems associated with sarcopenia will allow us to identify novel therapeutic interventions, such as omega-3 polyunsaturated fatty acids, to treat this disease.
    Keywords:  autophagy; docosahexaenoic acid (DHA); omega-3 polyunsaturated fatty acid (PUFA); proteasome; proteolysis; proteostasis; sarcopenia; ubiquitin
    DOI:  https://doi.org/10.3390/nu12092597
  54. Cancer Sci. 2020 Aug 28.
    Chaperon-Mediated Autophagy Receptor Modulates Tumor Growth and Chemoresistance in Non-Small Cell Lung Cancer.
    Akihiro Ichikawa, Yu Fujita, Yusuke Hosaka, Tsukasa Kadota, Akihiko Ito, Shigehiro Yagishita, Naoaki Watanabe, Shota Fujimoto, Hironori Kawamoto, Nayuta Saito, Masahiro Yoshida, Mitsuo Hashimoto, Shunsuke Minagawa, Hiromichi Hara, Noriko Motoi, Yusuke Yamamoto, Takahiro Ochiya, Jun Araya, Kazuyoshi Kuwano.
      Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of selective soluble proteins. LAMP2A is the key receptor protein of CMA, downregulation of LAMP2A leads to CMA blockade. Although CMA activation has been involved in cancer growth, CMA status and functions in non-small cell lung cancer (NSCLC) by focusing on the roles in regulating chemosensitivity remain to be clarified. In this study, we found that elevated expression of LAMP2A is found in NSCLC cell lines as well as patient's tumors, and confers poor survival and platinum-resistance in NSCLC patients. LAMP2A knockdown in NSCLC cells suppressed cell proliferation and colony formation, and increased the sensitivity to chemotherapeutic drugs in vitro. Furthermore, we found that intrinsic apoptosis signaling is the mechanism of cell death involved with CMA blockade. Remarkably, LAMP2A knockdown repressed tumorigenicity and sensitized the tumors to cisplatin treatment in NSCLC-bearing mice. Our discoveries suggest that LAMP2A is involved in the regulation of cancer malignant phenotypes and represents a promising new target against chemoresistant NSCLC.
    Keywords:  Apoptosis; Chaperone-mediated autophagy; Chemoresistance; LAMP2A; Lung cancer
    DOI:  https://doi.org/10.1111/cas.14629
  55. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Aug 20. pii: S1388-1981(20)30197-9. [Epub ahead of print] 158805
    An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy.
    Anna Maria Giudetti, Flora Guerra, Serena Longo, Raffaella Beli, Roberta Romano, Fiore Manganelli, Maria Nolano, Vincenzo Mangini, Lucio Santoro, Cecilia Bucci.
      Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations.
    Keywords:  RAB7A; de novo lipogenesis; fibroblast; lipid droplet; lipid metabolism; neurodegenerative disease
    DOI:  https://doi.org/10.1016/j.bbalip.2020.158805
  56. Biochem Biophys Res Commun. 2020 Sep 10. pii: S0006-291X(20)31378-4. [Epub ahead of print]530(1): 301-306
    Class II Arfs require a brefeldin-A-sensitive factor for Golgi association.
    Selma Y Dejgaard, John F Presley.
      Arf proteins are small Ras-family GTPases which recruit clathrin and COPI coats to Golgi membranes and regulate components of the membrane trafficking machinery. It is believed membrane association and activity of Arfs is coupled to GTP binding, with GTP hydrolysis required for vesicle uncoating. In humans, four Arf proteins (Arf1, Arf3, Arf4 and Arf5) are Golgi-associated. Conflicting reports have suggested that HA-GFP-tagged Class II ARFs (Arf4 and Arf5) are recruited to membrane independently of the brefeldin A sensitive exchange factor GBF1, suggesting regulation fundamentally different from the Class I Arfs (Arf1, Arf3), or alternately that the GTPase cycle of GFP-tagged Class II Arfs is similar to other Arfs. We show that these results depend on the fluorescent tag, with Arf4-HA-GFP tag resistant to brefeldin, but Arf4-GFP acting similarly to Arf1-GFP in brefeldin-sensitivity and photobleach assays. Arf4-HA-GFP could be partially reverted to the behavior of Arf4-GFP by mutation of two aspartic acids in the HA tag to alanine. Our results, which indicate a high sensitivity of Arf4 to tagging, can explain the discrepancies between previous studies. We discuss the implications of this study for future work with tagged Arfs.
    Keywords:  Arf4; Brefeldin; Class II Arfs; FRAP; GFP; Golgi
    DOI:  https://doi.org/10.1016/j.bbrc.2020.07.001
  57. Sci Adv. 2020 Aug;6(33): eabb8771
    TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals.
    Hua Jin, Weijin Xu, Reazur Rahman, Daxiang Na, Allegra Fieldsend, Wei Song, Shaobo Liu, Chong Li, Michael Rosbash.
      4E-BP (eIF4E-BP) represses translation initiation by binding to the 5' cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity, and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used HyperTRIBE (targets of RNA binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets more substantially compared to nontargets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.
    DOI:  https://doi.org/10.1126/sciadv.abb8771
  58. Int J Mol Sci. 2020 Aug 23. pii: E6074. [Epub ahead of print]21(17):
    The Endosomal Recycling Pathway-At the Crossroads of the Cell.
    Mary J O'Sullivan, Andrew J Lindsay.
      The endosomal recycling pathway lies at the heart of the membrane trafficking machinery in the cell. It plays a central role in determining the composition of the plasma membrane and is thus critical for normal cellular homeostasis. However, defective endosomal recycling has been linked to a wide range of diseases, including cancer and some of the most common neurological disorders. It is also frequently subverted by many diverse human pathogens in order to successfully infect cells. Despite its importance, endosomal recycling remains relatively understudied in comparison to the endocytic and secretory transport pathways. A greater understanding of the molecular mechanisms that support transport through the endosomal recycling pathway will provide deeper insights into the pathophysiology of disease and will likely identify new approaches for their detection and treatment. This review will provide an overview of the normal physiological role of the endosomal recycling pathway, describe the consequences when it malfunctions, and discuss potential strategies for modulating its activity.
    Keywords:  Rab GTPases; cancer; endosomal recycling pathway; neurological disorders; pathogen infection; plasma membrane; small molecule inhibitors; vesicle trafficking
    DOI:  https://doi.org/10.3390/ijms21176074
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