bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒11‒08
forty-three papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.
  2. The Emerging Role of the Lysosome in Parkinson's Disease.
  3. Autophagy-independent function of lipidated LC3 essential for TFEB activation during the lysosomal damage responses.
  4. TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles.
  5. Two-pore and TRPML cation channels: Regulators of phagocytosis, autophagy and lysosomal exocytosis.
  6. A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export.
  7. In vitro N-glycan mannosyl-phosphorylation of a therapeutic enzyme by using recombinant Mnn14 produced from Pichia pastoris.
  8. Target of Rapamycin in Control of Autophagy: Puppet Master and Signal Integrator.
  9. The Emerging and Diverse Roles of Bis(monoacylglycero) Phosphate Lipids in Cellular Physiology and Disease.
  10. β-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.
  11. Lysosomal Acid Lipase Drives Adipocyte Cholesterol Homeostasis and Modulates Lipid Storage in Obesity, Independent of Autophagy.
  12. TOR Complex 2- independent mutations in the regulatory PIF pocket of Gad8AKT1/SGK1 define separate branches of the stress response mechanisms in fission yeast.
  13. LRRC8 family proteins within lysosomes regulate cellular osmoregulation and enhance cell survival to multiple physiological stresses.
  14. Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors.
  15. Regulation of a Class III PI3K Lipid Kinase (VPS34 Complex II) by the Small G Protein Rab5A: Single Molecule Analysis Reveals a Dual Regulatory Mechanism.
  16. Cardiac pathology in Mucopolysaccharidosis I mice: Losartan modifies ERK1/2 activation during cardiac remodeling.
  17. Modeling Mucopolysaccharidosis Type II in the Fruit Fly by Using the RNA Interference Approach.
  18. The role of brain innate immune response in lysosomal storage disorders: fundamental process or evolutionary side-effect?
  19. Functional complementation of V-ATPase a subunit isoforms in osteoclasts.
  20. The 3.2-Å resolution structure of human mTORC2.
  21. Inositol-requiring enzyme-1 regulates phosphoinositide signaling lipids and macrophage growth.
  22. Therapeutic advances in Fabry disease: The future awaits.
  23. Therapeutic Options for Mucopolysaccharidosis II (Hunter Disease).
  24. Enhanced efficacy of JAK1 inhibitor with mTORC1/C2 targeting in smoldering/chronic adult T cell leukemia.
  25. mTORC1 activation in lung mesenchyme drives sex- and age-dependent pulmonary structure and function decline.
  26. GDC-0349 inhibits non-small cell lung cancer cell growth.
  27. White matter integrity correlates with cognition and disease severity in Fabry disease.
  28. Translational control in the naked mole-rat as a model highly resistant to cancer.
  29. Effects of rapamycin and AZD3463 combination on apoptosis, autophagy, and cell cycle for resistance control in breast cancer.
  30. Phosphatidylinositol-3-OH kinase signalling is spatially organized at endosomal compartments by microtubule-associated protein 4.
  31. Eukaryotic translation initiation factors as promising targets in cancer therapy.
  32. The Polyphenol Pterostilbene Ameliorates the Myopathic Phenotype of Collagen VI Deficient Mice via Autophagy Induction.
  33. Current Insights in Elucidation of Possible Molecular Mechanisms of the Juvenile Form of Batten Disease.
  34. The therapeutic value of XL388 in human glioma cells.
  35. The dialogue between the ubiquitin-proteasome system and autophagy: implications in ageing.
  36. Hip disease in Mucopolysaccharidoses and Mucolipidoses: A review of mechanisms, interventions and future perspectives.
  37. Bafilomycin A1 enhances NLRP3 inflammasome activation in human monocytes independent of lysosomal acidification.
  38. ER associated degradation preserves hematopoietic stem cell quiescence and self-renewal by restricting mTOR activity.
  39. Combined treatment of mitoxantrone sensitizes breast cancer cells to rapalogs through blocking eEF-2K-mediated activation of Akt and autophagy.
  40. Targeting Metabolic Plasticity and Flexibility Dynamics for Cancer Therapy.
  41. Gβγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils.
  42. Fabry disease: GLA deletion alters a canonical splice site in a family with neuropsychiatric manifestations.
  43. Fluorescent Probes for Super-Resolution Microscopy of Lysosomes.
  1. Mol Cell. 2020 Nov 05. pii: S1097-2765(20)30687-0. [Epub ahead of print]80(3): 437-451.e6
    Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.
    Yao Yao, Sungki Hong, Takayuki Ikeda, Hiroyuki Mori, Ormond A MacDougald, Shigeyuki Nada, Masato Okada, Ken Inoki.
      Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.
    Keywords:  ATXN3; Rag; Ragulator; Rheb; amino acids; deubiquitination; lysosome; mTORC1; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2020.10.004
  2. Cells. 2020 Nov 02. pii: E2399. [Epub ahead of print]9(11):
    The Emerging Role of the Lysosome in Parkinson's Disease.
    Alba Navarro-Romero, Marta Montpeyó, Marta Martinez-Vicente.
      Lysosomal function has a central role in maintaining neuronal homeostasis, and, accordingly, lysosomal dysfunction has been linked to neurodegeneration and particularly to Parkinson's disease (PD). Lysosomes are the converging step where the substrates delivered by autophagy and endocytosis are degraded in order to recycle their primary components to rebuild new macromolecules. Genetic studies have revealed the important link between the lysosomal function and PD; several of the autosomal dominant and recessive genes associated with PD as well as several genetic risk factors encode for lysosomal, autophagic, and endosomal proteins. Mutations in these PD-associated genes can cause lysosomal dysfunction, and since α-synuclein degradation is mostly lysosomal-dependent, among other consequences, lysosomal impairment can affect α-synuclein turnover, contributing to increase its intracellular levels and therefore promoting its accumulation and aggregation. Recent studies have also highlighted the bidirectional link between Parkinson's disease and lysosomal storage diseases (LSD); evidence includes the presence of α-synuclein inclusions in the brain regions of patients with LSD and the identification of several lysosomal genes involved in LSD as genetic risk factors to develop PD.
    Keywords:  Parkinson’s disease; autophagy; endocytosis; glucocerebrosidase; lysosomal storage diseases; lysosomes; α-synuclein
    DOI:  https://doi.org/10.3390/cells9112399
  3. Autophagy. 2020 Nov 05.
    Autophagy-independent function of lipidated LC3 essential for TFEB activation during the lysosomal damage responses.
    Shuhei Nakamura, Shiori Akayama, Tamotsu Yoshimori.
      Lysosomes are digestive organelles in cells containing many hydrolases, and also serve as a signaling hub to integrate intracellular and extracellular inputs; therefore, the integrity of lysosomes is critical for cellular homeostasis. Many agents and conditions can damage lysosomal membranes, which lead to leakage of lysosomal acidic contents into the cytosol thus becoming harmful for cells. Accordingly, cells have developed several defense systems to cope with damaged lysosomes, but underlying mechanisms of each system and their cross-talks are unclear. In our recent study, we found that a master transcription factor regulating autophagy and lysosomal biogenesis, TFEB (transcription factor EB) is activated during lysosomal damage, and this activation depends on an autophagy-independent function of lipidated LC3, which localizes on lysosomes. We further showed that this regulatory mechanism is essential to prevent the progression of the crystal nephropathy that accompanies lysosomal damage.
    Keywords:  Autophagy; LC3; TFEB; TRPML1; lysosome
    DOI:  https://doi.org/10.1080/15548627.2020.1846292
  4. Curr Biol. 2020 Oct 30. pii: S0960-9822(20)31530-X. [Epub ahead of print]
    TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles.
    Zilei Chen, Pedro Carpio Malia, Riko Hatakeyama, Raffaele Nicastro, Zehan Hu, Marie-Pierre Péli-Gulli, Jieqiong Gao, Taki Nishimura, Elja Eskes, Christopher J Stefan, Joris Winderickx, Jörn Dengjel, Claudio De Virgilio, Christian Ungermann.
      Organelles of the endomembrane system maintain their identity and integrity during growth or stress conditions by homeostatic mechanisms that regulate membrane flux and biogenesis. At lysosomes and endosomes, the Fab1 lipid kinase complex and the nutrient-regulated target of rapamycin complex 1 (TORC1) control the integrity of the endolysosomal homeostasis and cellular metabolism. Both complexes are functionally connected as Fab1-dependent generation of PI(3,5)P2 supports TORC1 activity. Here, we identify Fab1 as a target of TORC1 on signaling endosomes, which are distinct from multivesicular bodies, and provide mechanistic insight into their crosstalk. Accordingly, TORC1 can phosphorylate Fab1 proximal to its PI3P-interacting FYVE domain, which causes Fab1 to shift to signaling endosomes, where it generates PI(3,5)P2. This, in turn, regulates (1) vacuole morphology, (2) recruitment of TORC1 and the TORC1-regulatory Rag GTPase-containing EGO complex to signaling endosomes, and (3) TORC1 activity. Thus, our study unravels a regulatory feedback loop between TORC1 and the Fab1 complex that controls signaling at endolysosomes.
    Keywords:  Fab1; PI(3,5)P2; TORC1; cellular signaling; late endosome; lipid kinase; lysosome; phosphoinositide; signaling endosome; vacuole
    DOI:  https://doi.org/10.1016/j.cub.2020.10.026
  5. Pharmacol Ther. 2020 Oct 24. pii: S0163-7258(20)30244-8. [Epub ahead of print] 107713
    Two-pore and TRPML cation channels: Regulators of phagocytosis, autophagy and lysosomal exocytosis.
    A Scotto Rosato, R Tang, C Grimm.
      The old Greek saying "Panta Rhei" ("everything flows") is true for all life and all living things in general. It also becomes nicely evident when looking closely into cells. There, material from the extracellular space is taken up by endocytic processes and transported to endosomes where it is sorted either for recycling or degradation. Cargo is also packaged for export through exocytosis involving the Golgi network, lysosomes and other organelles. Everything in this system is in constant motion and many proteins are necessary to coordinate transport along the different intracellular pathways to avoid chaos. Among these proteins are ion channels., in particular TRPML channels (mucolipins) and two-pore channels (TPCs) which reside on endosomal and lysosomal membranes to speed up movement between organelles, e.g. by regulating fusion and fission; they help readjust pH and osmolarity changes due to such processes, or they promote exocytosis of export material. Pathophysiologically, these channels are involved in neurodegenerative, metabolic, retinal and infectious diseases, cancer, pigmentation defects, and immune cell function, and thus have been proposed as novel pharmacological targets, e.g. for the treatment of lysosomal storage disorders, Duchenne muscular dystrophy, or different types of cancer. Here, we discuss the similarities but also differences of TPCs and TRPMLs in regulating phagocytosis, autophagy and lysosomal exocytosis, and we address the contradictions and open questions in the field relating to the roles TPCs and TRPMLs play in these different processes.
    Keywords:  Autophagy; Calcium; Endo-lysosomal cation channel; Endocytic pathway; Lysosomal exocytosis; MCOLN; Neurodegeneration; TPC; TPCN; TRPML
    DOI:  https://doi.org/10.1016/j.pharmthera.2020.107713
  6. Nat Commun. 2020 11 03. 11(1): 5559
    A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export.
    Dick J H van den Boomen, Agata Sienkiewicz, Ilana Berlin, Marlieke L M Jongsma, Daphne M van Elsland, J Paul Luzio, Jacques J C Neefjes, Paul J Lehner.
      Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.
    DOI:  https://doi.org/10.1038/s41467-020-19032-0
  7. J Microbiol Biotechnol. 2020 Oct 28.
    In vitro N-glycan mannosyl-phosphorylation of a therapeutic enzyme by using recombinant Mnn14 produced from Pichia pastoris.
    Ji-Yeon Kang, Hong-Yeol Choi, Dong-Il Kim, Ohsuk Kwon, Doo-Byoung Oh.
      Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 77 amino acids spanning the transmembrane domain (47 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bisform which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.
    Keywords:  Enzyme replacement therapy; Lysosomal storage disease; Mannose-6-phosphate; Mannosyl-phosphorylation; Mnn14
    DOI:  https://doi.org/10.4014/jmb.2010.10033
  8. Int J Mol Sci. 2020 Nov 04. pii: E8259. [Epub ahead of print]21(21):
    Target of Rapamycin in Control of Autophagy: Puppet Master and Signal Integrator.
    Yosia Mugume, Zakayo Kazibwe, Diane C Bassham.
      The target of rapamycin (TOR) is an evolutionarily-conserved serine/threonine kinase that senses and integrates signals from the environment to coordinate developmental and metabolic processes. TOR senses nutrients, hormones, metabolites, and stress signals to promote cell and organ growth when conditions are favorable. However, TOR is inhibited when conditions are unfavorable, promoting catabolic processes such as autophagy. Autophagy is a macromolecular degradation pathway by which cells degrade and recycle cytoplasmic materials. TOR negatively regulates autophagy through phosphorylation of ATG13, preventing activation of the autophagy-initiating ATG1-ATG13 kinase complex. Here we review TOR complex composition and function in photosynthetic and non-photosynthetic organisms. We also review recent developments in the identification of upstream TOR activators and downstream effectors of TOR. Finally, we discuss recent developments in our understanding of the regulation of autophagy by TOR in photosynthetic organisms.
    Keywords:  TOR signaling; TORC1; autophagy; plant growth; target of rapamycin (TOR)
    DOI:  https://doi.org/10.3390/ijms21218259
  9. Int J Mol Sci. 2020 Oct 29. pii: E8067. [Epub ahead of print]21(21):
    The Emerging and Diverse Roles of Bis(monoacylglycero) Phosphate Lipids in Cellular Physiology and Disease.
    Megan R Showalter, Anastasia L Berg, Alexander Nagourney, Hailey Heil, Kermit L Carraway, Oliver Fiehn.
      Although understudied relative to many phospholipids, accumulating evidence suggests that bis(monoacylglycero)phosphate (BMP) is an important class of regulatory lipid that plays key roles in lysosomal integrity and function. BMPs are rare in most mammalian tissues, comprising only a few percent of total cellular lipid content, but are elevated in cell types such as macrophages that rely heavily on lysosomal function. BMPs are markedly enriched in endosomal and lysosomal vesicles compared to other organelles and membranous structures, and their unique sn-1:sn-1' stereoconfiguration may confer stability within the hydrolytic lysosomal environment. BMP-enriched vesicles serve in endosomal-lysosomal trafficking and function as docking structures for the activation of lysosomal hydrolytic enzymes, notably those involved in the catabolic breakdown of sphingolipids. BMP levels are dysregulated in lysosomal storage disorders, phospholipidosis, metabolic diseases, liver and kidney diseases and neurodegenerative disorders. However, whether BMP alteration is a mediator or simply a marker of pathological states is unclear. Likewise, although BMP acyl chain composition may be altered with disease states, the functional significance of specific BMP species remains to be resolved. Newly developed tools for untargeted lipidomic analysis, together with a deeper understanding of enzymes mediating BMP synthesis and degradation, will help shed further light on the functional significance of BMPs in cellular physiology and pathology.
    Keywords:  bis(monoacylglycero)phosphate; lipidomics; lysobisphophatidic acid; lysosome
    DOI:  https://doi.org/10.3390/ijms21218067
  10. Cell. 2020 Oct 27. pii: S0092-8674(20)31446-X. [Epub ahead of print]
    β-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.
    Sourish Ghosh, Teegan A Dellibovi-Ragheb, Adeline Kerviel, Eowyn Pak, Qi Qiu, Matthew Fisher, Peter M Takvorian, Christopher Bleck, Victor W Hsu, Anthony R Fehr, Stanley Perlman, Sooraj R Achar, Marco R Straus, Gary R Whittaker, Cornelis A M de Haan, John Kehrl, Grégoire Altan-Bonnet, Nihal Altan-Bonnet.
      β-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that β-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of β-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. β-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.
    Keywords:  CD1067700; Rab7; SARS-CoV-2; acidification/deacidification ARL8b; antigen presentation; coronavirus; lysosome; pH; viral egress; viral immunology
    DOI:  https://doi.org/10.1016/j.cell.2020.10.039
  11. Diabetes. 2020 Nov 02. pii: db200578. [Epub ahead of print]
    Lysosomal Acid Lipase Drives Adipocyte Cholesterol Homeostasis and Modulates Lipid Storage in Obesity, Independent of Autophagy.
    Camille Gamblin, Christine Rouault, Amélie Lacombe, Francina Langa-Vives, Dominique Farabos, Antonin Lamaziere, Karine Clément, Emmanuel L Gautier, Laurent Yvan-Charvet, Isabelle Dugail.
      Besides cytoplasmic lipase-dependent adipocyte fat mobilization, the metabolic role of lysosomal acid lipase (LAL), highly expressed in adipocytes is unclear. We show that the isolated adipocyte fraction but not the total undigested adipose tissue from obese patients has decreased LAL expression compared to non-obese. Lentiviral-mediated LAL knockdown in 3T3L1 to mimic obese adipocytes condition did not affect lysosome density or autophagic flux, but increased triglyceride storage and disrupted ER cholesterol as indicated by activated SREBP. Conversely, mice with adipose-specific LAL overexpression (Adpn-rtTA x TetO-hLAL) gained less weight and body fat than controls on a high fat diet, resulting in ameliorated glucose tolerance. Blood cholesterol was lower than controls albeit similar triglyceridemia. Adipose-LAL overexpressing mice phenotype is dependent on the housing temperature, and develops only under mild hypothermic stress (room temperature) but not at thermoneutrality (30°C), demonstrating prominent contribution of BAT thermogenesis. LAL overexpression increased BAT free cholesterol, decreased SREBP targets, and induced the expression of genes involved in initial steps of mitochondrial steroidogenesis, suggesting conversion of lysosome-derived cholesterol to pregnenolone. In conclusion, our study demonstrates that adipose LAL drives tissue cholesterol homeostasis and impacts BAT metabolism, suggesting beneficial LAL activation in anti-obesity approaches aimed at reactivating thermogenic energy expenditure.
    DOI:  https://doi.org/10.2337/db20-0578
  12. PLoS Genet. 2020 Nov 02. 16(11): e1009196
    TOR Complex 2- independent mutations in the regulatory PIF pocket of Gad8AKT1/SGK1 define separate branches of the stress response mechanisms in fission yeast.
    Emese Pataki, LubFtablea Simhaev, Hamutal Engel, Adiel Cohen, Martin Kupiec, Ronit Weisman.
      The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast, Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes.
    DOI:  https://doi.org/10.1371/journal.pgen.1009196
  13. Proc Natl Acad Sci U S A. 2020 Nov 02. pii: 202016539. [Epub ahead of print]
    LRRC8 family proteins within lysosomes regulate cellular osmoregulation and enhance cell survival to multiple physiological stresses.
    Ping Li, Meiqin Hu, Ce Wang, Xinghua Feng, ZhuangZhuang Zhao, Ying Yang, Nirakar Sahoo, Mingxue Gu, Yexin Yang, Shiyu Xiao, Rajan Sah, Timothy L Cover, Janet Chou, Raif Geha, Fernando Benavides, Richard I Hume, Haoxing Xu.
      LRRC8 family proteins on the plasma membrane play a critical role in cellular osmoregulation by forming volume-regulated anion channels (VRACs) necessary to prevent necrotic cell death. We demonstrate that intracellular LRRC8 proteins acting within lysosomes also play an essential role in cellular osmoregulation. LRRC8 proteins on lysosome membranes generate large lysosomal volume-regulated anion channel (Lyso-VRAC) currents in response to low cytoplasmic ionic strength conditions. When a double-leucine L706L707 motif at the C terminus of LRRC8A was mutated to alanines, normal plasma membrane VRAC currents were still observed, but Lyso-VRAC currents were absent. We used this targeting mutant, as well as pharmacological tools, to demonstrate that Lyso-VRAC currents are necessary for the formation of large lysosome-derived vacuoles, which store and then expel excess water to maintain cytosolic water homeostasis. Thus, Lyso-VRACs allow lysosomes of mammalian cells to act as the cell`s "bladder." When Lyso-VRAC current was selectively eliminated, the extent of necrotic cell death to sustained stress was greatly increased, not only in response to hypoosmotic stress, but also to hypoxic and hypothermic stresses. Thus Lyso-VRACs play an essential role in enabling cells to mount successful homeostatic responses to multiple stressors.
    Keywords:  chloride channel; exocytosis; lysosome; osmoregulation; vacuolation
    DOI:  https://doi.org/10.1073/pnas.2016539117
  14. Cells. 2020 Oct 31. pii: E2384. [Epub ahead of print]9(11):
    Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors.
    Yuka Okusha, Manh Tien Tran, Mami Itagaki, Chiharu Sogawa, Takanori Eguchi, Tatsuo Okui, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto.
      Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). Rab11A GTPase, belonging to Rab11 subfamily representing the largest branch of Ras superfamily of small GTPases, has been identified as one of the crucial regulators of cell surface receptor recycling. Nevertheless, the regulatory role of Rab11A in osteoclast differentiation has been completely unknown. In this study, we found that Rab11A was strongly upregulated at a late stage of osteoclast differentiation derived from bone marrow-derived macrophages (BMMs) or RAW-D murine osteoclast precursor cells. Rab11A silencing promoted osteoclast formation and significantly increased the surface levels of c-fms and receptor activator of nuclear factor-κB (RANK) while its overexpression attenuated osteoclast formation and the surface levels of c-fms and RANK. Using immunocytochemical staining for tracking Rab11A vesicular localization, we observed that Rab11A was localized in early and late endosomes, but not lysosomes. Intriguingly, Rab11A overexpression caused the enhancement of fluorescent intensity and size-based enlargement of early endosomes. Besides, Rab11A overexpression promoted lysosomal activity via elevating the endogenous levels of a specific lysosomal protein, LAMP1, and two key lysosomal enzymes, cathepsins B and D in osteoclasts. More importantly, inhibition of the lysosomal activity by chloroquine, we found that the endogenous levels of c-fms and RANK proteins were enhanced in osteoclasts. From these observations, we suggest a novel function of Rab11A as a negative regulator of osteoclastogenesis mainly through (i) abolishing the surface abundance of c-fms and RANK receptors, and (ii) upregulating lysosomal activity, subsequently augmenting the degradation of c-fms and RANK receptors, probably via the axis of early endosomes-late endosomes-lysosomes in osteoclasts.
    Keywords:  NFATc-1; RANK; Rab11A; c-fms; osteoclast; vesicular transport
    DOI:  https://doi.org/10.3390/cells9112384
  15. Biophys J. 2020 Oct 30. pii: S0006-3495(20)30852-3. [Epub ahead of print]
    Regulation of a Class III PI3K Lipid Kinase (VPS34 Complex II) by the Small G Protein Rab5A: Single Molecule Analysis Reveals a Dual Regulatory Mechanism.
    Thomas C Buckles, Yohei Ohashi, Shirley Tremel, Stephen H McLaughlin, Els Pardon, Jan Steyaert, Moshe Gordon, Roger L Williams, Joseph J Falke.
      VPS34 Complex II (VPS34CII) is a 386 kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits, which together function as a prototypical Class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, PROPPINS) to the membrane surface where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. (i) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. (ii) In the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. The present study employs an in vitro single molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A appears to provide virtually perfect binary on-off switching and is required for VPS34CII membrane binding and PI3P production.
    DOI:  https://doi.org/10.1016/j.bpj.2020.10.028
  16. J Inherit Metab Dis. 2020 Nov 04.
    Cardiac pathology in Mucopolysaccharidosis I mice: Losartan modifies ERK1/2 activation during cardiac remodeling.
    Esteban Alberto Gonzalez, Santiago Alonso Tobar Leitão, Douglas Dos Santos Soares, Angela Maria Vicente Tavares, Roberto Giugliani, Guilherme Baldo, Ursula Matte.
      Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by mutations in the IDUA gene, that codifies the alpha-L-iduronidase enzyme, which deficiency leads to storage of glycosaminoglycans, with multiple clinical manifestations. One of the leading causes of death in MPS I patients are cardiac complications such as cardiac valve thickening, conduction abnormalities, myocardial dysfunction, and cardiac hypertrophy. The mechanism leading to cardiac dysfunction in MPS I is not entirely understood. In a previous study, we have demonstrated that losartan and propranolol improved the cardiac function in MPS I mice. Thus, we aimed to investigate whether the pathways influenced by these drugs may modulate the cardiac remodeling process in MPS I mice. According to our previous observation, losartan and propranolol restore the heart function, without altering valve thickness. MPS I mice presented reduced activation of AKT and ERK1/2, increased activity of cathepsins, but no alteration in metalloproteinase activity was observed. Animals treated with losartan showed a reduction in cathepsin activity and restored ERK1/2 activation. While both losartan and propranolol improved heart function, no mechanistic evidence was found for propranolol so far. Our results suggest that losartan or propranolol could be used to ameliorate the cardiac disease in MPS I and could be considered as adjuvant treatment candidates for therapy optimization. This article is protected by copyright. All rights reserved.
    Keywords:  AKT; ERK1/2; Mucopolysaccharidosis type I; cardiac disease; cardiac remodeling; losartan; propranolol
    DOI:  https://doi.org/10.1002/jimd.12327
  17. Life (Basel). 2020 Oct 30. pii: E263. [Epub ahead of print]10(11):
    Modeling Mucopolysaccharidosis Type II in the Fruit Fly by Using the RNA Interference Approach.
    Laura Rigon, Nicole Kucharowski, Franka Eckardt, Reinhard Bauer.
      Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder that occurs due to the deficit of the lysosomal enzyme iduronate 2-sulfatase (IDS) that leads to the storage of the glycosaminoglycan heparan- and dermatan-sulfate in all organs and tissues. It is characterized by important clinical features and the severe form presents with a heavy neurological involvement. However, almost nothing is known about the neuropathogenesis of MPS II. To address this issue, we developed a ubiquitous, neuronal, and glial-specific knockdown model in Drosophila melanogaster by using the RNA interference (RNAi) approach. Knockdown of the Ids/CG12014 gene resulted in a significant reduction of the Ids gene expression and enzymatic activity. However, glycosaminoglycan storage, survival, molecular markers (Atg8a, Lamp1, Rab11), and locomotion behavior were not affected. Even strongly reduced, IDS-activity was enough to prevent a pathological phenotype in a MPS II RNAi fruit fly. Thus, a Drosophila MPS II model requires complete abolishment of the enzymatic activity.
    Keywords:  Drosophila melanogaster; Hunter Syndrome; Mucopolysaccharidosis; RNA interference; fruit fly; lysosomal storage disorders
    DOI:  https://doi.org/10.3390/life10110263
  18. FEBS Lett. 2020 Oct 31.
    The role of brain innate immune response in lysosomal storage disorders: fundamental process or evolutionary side-effect?
    Einat B Vitner.
      Sphingolipidoses are diseases caused by mutations in genes responsible for sphingolipid degradation and thereby lead to sphingolipid accumulation. Most sphingolipidoses have a neurodegenerative manifestation characterized by innate immune activation in the brain. However, the role of the immune response in disease progression is ill-understood. In contrast to infectious diseases, immune activation is unable to eliminate the offending agent in sphingolipidoses resulting in ineffective, chronic inflammation. This paradox begs two fundamental questions: why has this immune response evolved in sphingolipidoses? What role does it play in disease progression? Here, starting from the observation that sphingolipids are elevated also in infectious diseases, I discuss the possibility that activation of the brain immune response by sphingolipids has evolved as a part of the immune response against pathogens and plays no major role in sphingolipidoses.
    Keywords:  Guillain-Barré syndrome; brain; infectious diseases; innate immune response; lysosomal storage diseases; neuroinflammation; sphingolipidoses; viral infection
    DOI:  https://doi.org/10.1002/1873-3468.13980
  19. J Biochem. 2020 Nov 02. pii: mvaa118. [Epub ahead of print]
    Functional complementation of V-ATPase a subunit isoforms in osteoclasts.
    Naomi Matsumoto, Mizuki Sekiya, Yasuyuki Fujimoto, Satoshi Haga, Ge-Hong Sun-Wada, Yoh Wada, Mayumi Nakanishi-Matsui.
      In osteoclasts, the a3 isoform of the proton-pumping V-ATPase plays essential roles in anterograde trafficking of secretory lysosomes and extracellular acidification required for bone resorption. This study examined functional complementation of the a isoforms by exogenously expressing the a1, a2, and a3 isoforms in a3-knockout (KO) osteoclasts. The expression levels of a1 and a2 in a3KO osteoclasts were similar, but lower than that of a3. a1 significantly localized to lysosomes, whereas a2 slightly did. On the other hand, a2 interacted with Rab7, a regulator of secretory lysosome trafficking in osteoclasts, more efficiently than a1. a1 partly complemented the functions of a3 in secretory lysosome trafficking and calcium phosphate resorption, while a2 partly complemented the former but not the latter function.
    Keywords:  Rab7; V-ATPase; a subunit isoform; osteoclast; secretory lysosome
    DOI:  https://doi.org/10.1093/jb/mvaa118
  20. Sci Adv. 2020 Nov;pii: eabc1251. [Epub ahead of print]6(45):
    The 3.2-Å resolution structure of human mTORC2.
    Alain Scaiola, Francesca Mangia, Stefan Imseng, Daniel Boehringer, Karolin Berneiser, Mitsugu Shimobayashi, Edward Stuttfeld, Michael N Hall, Nenad Ban, Timm Maier.
      The protein kinase mammalian target of rapamycin (mTOR) is the central regulator of cell growth. Aberrant mTOR signaling is linked to cancer, diabetes, and neurological disorders. mTOR exerts its functions in two distinct multiprotein complexes, mTORC1 and mTORC2. Here, we report a 3.2-Å resolution cryo-EM reconstruction of mTORC2. It reveals entangled folds of the defining Rictor and the substrate-binding SIN1 subunits, identifies the carboxyl-terminal domain of Rictor as the source of the rapamycin insensitivity of mTORC2, and resolves mechanisms for mTORC2 regulation by complex destabilization. Two previously uncharacterized small-molecule binding sites are visualized, an inositol hexakisphosphate (InsP6) pocket in mTOR and an mTORC2-specific nucleotide binding site in Rictor, which also forms a zinc finger. Structural and biochemical analyses suggest that InsP6 and nucleotide binding do not control mTORC2 activity directly but rather have roles in folding or ternary interactions. These insights provide a firm basis for studying mTORC2 signaling and for developing mTORC2-specific inhibitors.
    DOI:  https://doi.org/10.1126/sciadv.abc1251
  21. EMBO Rep. 2020 Nov 02. e51462
    Inositol-requiring enzyme-1 regulates phosphoinositide signaling lipids and macrophage growth.
    Syed Muhammad Hamid, Mevlut Citir, Erdem Murat Terzi, Ismail Cimen, Zehra Yildirim, Asli Ekin Dogan, Begum Kocaturk, Umut Inci Onat, Moshe Arditi, Christian Weber, Alexis Traynor-Kaplan, Carsten Schultz, Ebru Erbay.
      The ER-bound kinase/endoribonuclease (RNase), inositol-requiring enzyme-1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1's one known, specific RNA target, X box-binding protein-1 (XBP1) or the RNA substrates of IRE1-dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide-derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross-talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1's RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P3 ) 5-phosphatase-2 (INPPL1) is a direct target of miR-2137, which controls PI(3,4,5)P3 levels in macrophages. The modulation of cellular PI(3,4,5)P3 /PIP2 ratio and anabolic mTOR signaling by the IRE1-induced miR-2137 demonstrates how the ER can provide a critical input into cell growth decisions.
    Keywords:  ER stress; hyperlipidemia; mTOR signaling; macrophage; microRNA
    DOI:  https://doi.org/10.15252/embr.202051462
  22. Biomed Pharmacother. 2020 Nov;pii: S0753-3322(20)30972-0. [Epub ahead of print]131 110779
    Therapeutic advances in Fabry disease: The future awaits.
    Sam Kant, Mohamed G Atta.
      Fabry disease (FD) is an X-linked disorder caused by mutations in GLA gene responsible for coding of the lysosomal enzyme alpha-galactosidase A(α-GAL). The resultant accumulation of globotriaosylceramide (Gb-3) leads to multisystemic disease including progressive chronic kidney disease, hypertrophic cardiomyopathy, stroke, angiokeratomas and corneal whorls. Current treatments include enzyme replacement therapy (ERT), along with recent advent of chaperone therapy. ERT has not shown to have dramatic improvement in outcomes for all organ systems, with benefit mostly seen in kidney disease and reduction in left ventricular hypertrophy. ERT, however, is associated with formation of anti-drug antibodies and requirement of long-term venous access, while chaperone therapy can only be used in amenable mutations. A multitude of therapies are now under investigation in various phases of clinical trials. These include pegylated form of α-GAL (pegunigalsidase alpha), gene therapy (both in-vivo and ex-vivo methods), mRNA therapy (inducing production of α-GAL) and substrate reduction therapy (inhibitors of glucosylceramide synthase leading to reduction of Gb-3). This review encapsulates literature pertaining to current and investigational therapies for FD.
    Keywords:  Chaperone therapy; Enzyme replacement therapy; Fabry disease; Gene therapy; Globotriaosylceramide; Substrate reduction therapy
    DOI:  https://doi.org/10.1016/j.biopha.2020.110779
  23. Curr Pharm Des. 2020 Jul 24.
    Therapeutic Options for Mucopolysaccharidosis II (Hunter Disease).
    Francyne Kubaski, Filippo Vairo, Guilherme Baldo, Fabiano de Oliveira Poswar, Amauri Dalla Corte, Roberto Giugliani.
      BACKGROUND: Mucopolysaccharidosis type II (Hunter syndrome, or MPS II) is an X-linked lysosomal disorder caused by the deficiency of iduronate-2-sulfatase, which leads to the accumulation of glycosaminoglycans (GAGs) in a variety of tissues, resulting in a multisystemic disease that can also impair the central nervous system (CNS).OBJECTIVE: This review focuses on providing the latest information and expert opinion about the therapies available and under development for MPS II.
    METHODS: We have comprehensively revised the latest studies about hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT - intravenous, intrathecal, intracerebroventricular, and intravenous with fusion proteins), small molecules, gene therapy/genome editing, and supportive management.
    RESULTS AND DISCUSSION: Intravenous ERT is a well-established specific therapy, which ameliorates the somatic features but not the CNS manifestations. Intrathecal or intracerebroventricular ERT and intravenous ERT with fusion proteins, presently under development, seem to be able to reduce the levels of GAGs in the CNS and have the potential of reducing the impact of the neurological burden of the disease. Gene therapy and/or genome editing have shown promising results in preclinical studies, bringing hope for a "one-time therapy" soon. Results with HSCT in MPS II are controversial, and small molecules could potentially address some disease manifestations. In addition to the specific therapeutic options, supportive care plays a major role in the management of these patients.
    CONCLUSION: At this time, the treatment of individuals with MPS II is mainly based on intravenous ERT whereas HSCT can be a potential alternative in specific cases. In the coming years, several new therapy options that target the neurological phenotype of MPS II should be available.
    Keywords:  Hunter syndrome; Mucopolysaccharidosis; enzyme replacement therapy; gene therapy; glycosaminoglycans; hematopoietic stem cell transplantation; iduronate-2-sulfatase
    DOI:  https://doi.org/10.2174/1381612826666200724161504
  24. Transl Oncol. 2020 Oct 22. pii: S1936-5233(20)30405-8. [Epub ahead of print]14(1): 100913
    Enhanced efficacy of JAK1 inhibitor with mTORC1/C2 targeting in smoldering/chronic adult T cell leukemia.
    Anusara Daenthanasanmak, Yuquan Lin, Meili Zhang, Bonita R Bryant, Michael N Petrus, Richard N Bamford, Craig J Thomas, Milos D Miljkovic, Kevin C Conlon, Thomas A Waldmann.
      Adult T-cell leukemia (ATL) is an aggressive T-cell lymphoproliferative malignancy of regulatory T lymphocytes (Tregs), caused by human T-cell lymphotropic virus 1 (HTLV-1). Interleukin 2 receptor alpha (IL-2Rα) is expressed in the leukemic cells of smoldering/chronic ATL patients, leading to constitutive activation of the JAK/STAT pathway and spontaneous proliferation. The PI3K/AKT/mTOR pathway also plays a critical role in ATL cell survival and proliferation. We previously performed a high-throughput screen that demonstrated additive/synergistic activity of Ruxolitinib, a JAK1/2 inhibitor, with AZD8055, an mTORC1/C2 inhibitor. However, effects of unintended JAK2 inhibition with Ruxolitinib limits it therapeutic potential for ATL patients, which lead us to evaluate a JAK1-specific inhibitor. Here, we demonstrated that Upadacitinib, a JAK-1 inhibitor, inhibited the proliferation of cytokine-dependent ATL cell lines and the expression of p-STAT5. Combinations of Upadacitinib with either AZD8055 or Sapanisertib, mTORC1/C2 inhibitors, showed anti-proliferative effects against cytokine-dependent ATL cell lines and synergistic effect with reducing tumor growth in NSG mice bearing IL-2 transgenic tumors. Importantly, the combination of these two agents inhibited ex vivo spontaneous proliferation of ATL cells from patients with smoldering/chronic ATL. Combined targeting of JAK/STAT and PI3K/AKT/mTOR pathways represents a promising therapeutic intervention for patients with smoldering/chronic ATL.
    Keywords:  Adult T cell leukemia; Combination therapy; JAK1 inhibitors; Smoldering/chronic ATL; mTOR inhibitors
    DOI:  https://doi.org/10.1016/j.tranon.2020.100913
  25. Nat Commun. 2020 Nov 06. 11(1): 5640
    mTORC1 activation in lung mesenchyme drives sex- and age-dependent pulmonary structure and function decline.
    Kseniya Obraztsova, Maria C Basil, Ryan Rue, Aravind Sivakumar, Susan M Lin, Alexander R Mukhitov, Andrei I Gritsiuta, Jilly F Evans, Meghan Kopp, Jeremy Katzen, Annette Robichaud, Elena N Atochina-Vasserman, Shanru Li, Justine Carl, Apoorva Babu, Michael P Morley, Edward Cantu, Michael F Beers, David B Frank, Edward E Morrisey, Vera P Krymskaya.
      Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/TSC2) genes coding for suppressors of the mechanistic target of rapamycin complex 1 (mTORC1). The origin of LAM cells is still unknown. Here, we profile a LAM lung compared to an age- and sex-matched healthy control lung as a hypothesis-generating approach to identify cell subtypes that are specific to LAM. Our single-cell RNA sequencing (scRNA-seq) analysis reveals novel mesenchymal and transitional alveolar epithelial states unique to LAM lung. This analysis identifies a mesenchymal cell hub coordinating the LAM disease phenotype. Mesenchymal-restricted deletion of Tsc2 in the mouse lung produces a mTORC1-driven pulmonary phenotype, with a progressive disruption of alveolar structure, a decline in pulmonary function, increase of rapamycin-sensitive expression of WNT ligands, and profound female-specific changes in mesenchymal and epithelial lung cell gene expression. Genetic inactivation of WNT signaling reverses age-dependent changes of mTORC1-driven lung phenotype, but WNT activation alone in lung mesenchyme is not sufficient for the development of mouse LAM-like phenotype. The alterations in gene expression are driven by distinctive crosstalk between mesenchymal and epithelial subsets of cells observed in mesenchymal Tsc2-deficient lungs. This study identifies sex- and age-specific gene changes in the mTORC1-activated lung mesenchyme and establishes the importance of the WNT signaling pathway in the mTORC1-driven lung phenotype.
    DOI:  https://doi.org/10.1038/s41467-020-18979-4
  26. Cell Death Dis. 2020 Nov 05. 11(11): 951
    GDC-0349 inhibits non-small cell lung cancer cell growth.
    Han Yang, Jun Zhao, Mengjing Zhao, Lihao Zhao, Li-Na Zhou, Yuxia Duan, Gang Li.
      Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related human mortality with a clear need for new therapeutic intervention. GDC-0349 is a potent and selective ATP-competitive mTOR inhibitor. In A549 cells and primary human NSCLC cells, GDC-0349 inhibited cell growth, proliferation, cell cycle progression, migration and invasion, while inducing significant apoptosis activation. Although GDC-0349 blocked Akt-mTORC1/2 activation in NSCLC cells, it also exerted cytotoxicity in Akt1-knockout A549 cells. Furthermore, restoring Akt-mTOR activation by a constitutively-active Akt1 only partially attenuated GDC-0349-induced A549 cell apoptosis, indicating the existence of Akt-mTOR-independent mechanisms. In NSCLC cells GDC-0349 induced sphingosine kinase 1 (SphK1) inhibition, ceramide accumulation, JNK activation and oxidative injury. Conversely, N-acetylcysteine, the JNK inhibitor and sphingosine 1-phosphate alleviated GDC-0349-induced NSCLC cell apoptosis. In vivo, daily oral administration of GDC-0349 potently inhibited NSCLC xenograft growth in mice. Akt-mTOR in-activation, SphK1 inhibition, JNK activation and oxidative stress were detected in NSCLC xenograft tissues with GDC-0349 administration. In summary, GDC-0349 inhibits NSCLC cell growth via Akt-mTOR-dependent and Akt-mTOR-independent mechanisms.
    DOI:  https://doi.org/10.1038/s41419-020-03146-w
  27. Brain. 2020 Nov 03. pii: awaa282. [Epub ahead of print]
    White matter integrity correlates with cognition and disease severity in Fabry disease.
    Leonardo Ulivi, Baris Kanber, Ferran Prados, Indran Davagnanam, Aine Merwick, Edgar Chan, Fay Williams, Derralynn Hughes, Elaine Murphy, R H Lachmann, Claudia A M Gandini Wheeler-Kingshott, Lisa Cipolotti, David J Werring.
      Cerebral white matter pathology is a common CNS manifestation of Fabry disease, visualized as white matter hyperintensities on MRI in 42-81% of patients. Diffusion tensor imaging (DTI) MRI is a sensitive technique to quantify microstructural damage within the white matter with potential value as a disease biomarker. We evaluated the pattern of DTI abnormalities in Fabry disease, and their correlations with cognitive impairment, mood, anxiety, disease severity and plasma lyso-Gb3 levels in 31 patients with genetically proven Fabry disease and 19 age-matched healthy control subjects. We obtained average values of fractional anisotropy and mean diffusivity within the white matter and performed voxelwise analysis with tract-based spatial statistics. Using a standardized neuropsychological test battery, we assessed processing speed, executive function, anxiety, depression and disease severity. The mean age (% male) was 44.1 (45%) for patients with Fabry disease and 37.4 (53%) for the healthy control group. In patients with Fabry disease, compared to healthy controls the mean average white matter fractional anisotropy was lower in [0.423 (standard deviation, SD 0.023) versus 0.446 (SD 0.016), P = 0.002] while mean average white matter mean diffusivity was higher (749 × 10-6 mm2/s (SD 32 × 10-6) versus 720 × 10-6 mm2/s (SD 21 × 10-6), P = 0.004]. Voxelwise statistics showed that the diffusion abnormalities for both fractional anisotropy and mean diffusivity were anatomically widespread. A lesion probability map showed that white matter hyperintensities also had a wide anatomical distribution with a predilection for the posterior centrum semiovale. However, diffusion abnormalities in Fabry disease were not restricted to lesional tissue; compared to healthy controls, the normal appearing white matter in patients with Fabry disease had reduced fractional anisotropy [0.422 (SD 0.022) versus 0.443 (SD 0.017) P = 0.003] and increased mean diffusivity [747 × 10-6 mm2/s (SD 26 × 10-6) versus 723 × 10-6 mm2/s (SD 22 × 10-6), P = 0.008]. Within patients, average white matter fractional anisotropy and white matter lesion volume showed statistically significant correlations with Digit Symbol Coding Test score (r = 0.558, P = 0.001; and r = -0.633, P ≤ 0.001, respectively). Average white matter fractional anisotropy correlated with the overall Mainz Severity Score Index (r = -0.661, P ≤ 0.001), while average white matter mean diffusivity showed a strong correlation with plasma lyso-Gb3 levels (r = 0.559, P = 0.001). Our findings using DTI confirm widespread areas of microstructural white matter disruption in Fabry disease, extending beyond white matter hyperintensities seen on conventional MRI. Moreover, diffusion measures show strong correlations with cognition (processing speed), clinical disease severity and a putative plasma biomarker of disease activity, making them promising quantitative biomarkers for monitoring Fabry disease severity and progression.
    Keywords:  Fabry disease; central nervous system; cognition; diffusion tensor imaging; white matter pathology
    DOI:  https://doi.org/10.1093/brain/awaa282
  28. Biochim Biophys Acta Rev Cancer. 2020 Oct 22. pii: S0304-419X(20)30174-8. [Epub ahead of print]1875(1): 188455
    Translational control in the naked mole-rat as a model highly resistant to cancer.
    Giovanna Maldonado, Greco Hernández.
      Dysregulation of mRNA translation is involved in the onset and progression of different types of cancer. To gain insight into novel genetic strategies to avoid this malady, we reviewed the available genomic, transcriptomic, and proteomic data about the translational machinery from the naked-mole rat (NMR) Heterocephalus glaber, a new model of study that exhibits high resistance to cancer. The principal features that might confer cancer resistance are 28S rRNA fragmentation, RPL26 and eIF4G overexpression, global downregulation of mTOR pathway, specific amino acid residues in RAPTOR (P908) and RICTOR (V1695), and the absence of 4E-BP3. These features are not only associated with cancer but also might couple longevity and adaptation to hypoxia. We propose that the regulation of translation is among the strategies endowing NMR cancer resistance.
    Keywords:  Cancer; Heterocephalus glaber; Hypoxia; Naked mole-rat; Translational control; mTOR
    DOI:  https://doi.org/10.1016/j.bbcan.2020.188455
  29. Life Sci. 2020 Oct 24. pii: S0024-3205(20)31396-5. [Epub ahead of print] 118643
    Effects of rapamycin and AZD3463 combination on apoptosis, autophagy, and cell cycle for resistance control in breast cancer.
    Neslihan Pınar Ozates, Fatma Soğutlu, Ferzan Lerminoglu, Busra Demir, Cumhur Gunduz, Behrouz Shademan, Cigir Biray Avci.
      Breast cancer is the most common cancer in women and the leading cause of cancer mortality in women over 40 it's the year. The existence of the PI3K/AKT/mTOR pathway aberrations in more than 70% of breast cancer has caused to become a therapeutic target. AZD3463 is an anti-cancer agent used as a potential inhibitor of ALK/IGF1R. It also induces apoptosis and autophagy of the PI3K/AKT/mTOR pathway in cancer cells. Although the mTOR signaling might be inhibited by rapamycin treatment, signals transmitted from the upstream pathway supports cell survival and proliferation. The WST-1 assay test was performed to evaluate the anti-proliferative effects of rapamycin and AZD3463. Besides, the effects of them on apoptosis, autophagy, cytostatic, and metabolism in MCF7 breast cancer cells were investigated. Also, changes in the expression of apoptotic regulatory genes, cell cycle, and metabolism in the PI3K/AKT/mTOR Pathway were determined by Quantitative RT-PCR. The results showed that rapamycin and AZD3463 treatments significantly reduced survival in MCF7 cells. Also, apoptosis, autophagy, and cell population in the G0/G1 stage in the MCF7 cell category in the treatment group showed an increase compared to the control group. The combination of rapamycin and AZD3463 (AZD-RAPA) was determined as an additive according to isobologram analysis. In the combination of rapamycin with AZD3463, the expression of CDKN1B, PTEN, FOXO3, and APC genes increases, and the expression of PRKCB and PIK3CG genes decreases. Our results showed that the use of AZD-RAPA reduced the resistance of cancer cells to treatment and it leads cancer cells to apoptosis.
    Keywords:  Autophagy; Breast cancer; Drug resistance; Rapamycin; mTOR signaling pathways
    DOI:  https://doi.org/10.1016/j.lfs.2020.118643
  30. Nat Cell Biol. 2020 Nov;22(11): 1357-1370
    Phosphatidylinositol-3-OH kinase signalling is spatially organized at endosomal compartments by microtubule-associated protein 4.
    Narendra Thapa, Mo Chen, Hudson T Horn, Suyong Choi, Tianmu Wen, Richard A Anderson.
      The canonical model of agonist-stimulated phosphatidylinositol-3-OH kinase (PI3K)-Akt signalling proposes that PI3K is activated at the plasma membrane, where receptors are activated and phosphatidylinositol-4,5-bisphosphate is concentrated. Here we show that phosphatidylinositol-3,4,5-trisphosphate generation and activated Akt are instead largely confined to intracellular membranes upon receptor tyrosine kinase activation. Microtubule-associated protein 4 (MAP4) interacts with and controls localization of membrane vesicle-associated PI3Kα to microtubules. The microtubule-binding domain of MAP4 binds directly to the C2 domain of the p110α catalytic subunit. MAP4 controls the interaction of PI3Kα with activated receptors at endosomal compartments along microtubules. Loss of MAP4 results in the loss of PI3Kα targeting and loss of PI3K-Akt signalling downstream of multiple agonists. The MAP4-PI3Kα assembly defines a mechanism for spatial control of agonist-stimulated PI3K-Akt signalling at internal membrane compartments linked to the microtubule network.
    DOI:  https://doi.org/10.1038/s41556-020-00596-4
  31. Cell Commun Signal. 2020 Nov 04. 18(1): 175
    Eukaryotic translation initiation factors as promising targets in cancer therapy.
    Peiqi Hao, Jiaojiao Yu, Richard Ward, Yin Liu, Qiao Hao, Su An, Tianrui Xu.
      The regulation of the translation of messenger RNA (mRNA) in eukaryotic cells is critical for gene expression, and occurs principally at the initiation phase which is mainly regulated by eukaryotic initiation factors (eIFs). eIFs are fundamental for the translation of mRNA and as such act as the primary targets of several signaling pathways to regulate gene expression. Mis-regulated mRNA expression is a common feature of tumorigenesis and the abnormal activity of eIF complexes triggered by upstream signaling pathways is detected in many tumors, leading to the selective translation of mRNA encoding proteins involved in tumorigenesis, metastasis, or resistance to anti-cancer drugs, and making eIFs a promising therapeutic target for various types of cancers. Here, we briefly outline our current understanding of the biology of eIFs, mainly focusing on the effects of several signaling pathways upon their functions and discuss their contributions to the initiation and progression of tumor growth. An overview of the progress in developing agents targeting the components of translation machinery for cancer treatment is also provided. Video abstract.
    Keywords:  Cancer; MAPK; PI3K/Akt; eIF; mRNA translation; mTOR
    DOI:  https://doi.org/10.1186/s12964-020-00607-9
  32. Front Cell Dev Biol. 2020 ;8 580933
    The Polyphenol Pterostilbene Ameliorates the Myopathic Phenotype of Collagen VI Deficient Mice via Autophagy Induction.
    Samuele Metti, Lisa Gambarotto, Martina Chrisam, Martina Baraldo, Paola Braghetta, Bert Blaauw, Paolo Bonaldo.
      The induction of autophagy, the catabolic pathway by which damaged or unnecessary cellular components are subjected to lysosome-mediated degradation and recycling, is impaired in Collagen VI (COL6) null mice and COL6-related myopathies. This autophagic impairment causes an accumulation of dysfunctional mitochondria, which in turn leads to myofiber degeneration. Our previous work showed that reactivation of autophagy in COL6-related myopathies is beneficial for muscle structure and function both in the animal model and in patients. Here we show that pterostilbene (Pt)-a non-toxic polyphenol, chemically similar to resveratrol but with a higher bioavailability and metabolic stability-strongly promotes in vivo autophagic flux in the skeletal muscle of both wild-type and COL6 null mice. Reactivation of autophagy in COL6-deficient muscles was also paralleled by several beneficial effects, including significantly decreased incidence of spontaneous apoptosis, recovery of ultrastructural defects and muscle remodeling. These findings point at Pt as an effective autophagy-inducing nutraceutical for skeletal muscle with great potential in counteracting the major pathogenic hallmarks of COL6-related myopathies, a valuable feature that may be also beneficial in other muscle pathologies characterized by defective regulation of the autophagic machinery.
    Keywords:  Collagen VI; autophagy; congenital muscular dystrophies; muscle remodeling; nutraceutical agent; skeletal muscle
    DOI:  https://doi.org/10.3389/fcell.2020.580933
  33. Int J Mol Sci. 2020 Oct 29. pii: E8055. [Epub ahead of print]21(21):
    Current Insights in Elucidation of Possible Molecular Mechanisms of the Juvenile Form of Batten Disease.
    Elena K Shematorova, George V Shpakovski.
      The neuronal ceroid lipofuscinoses (NCLs) collectively constitute one of the most common forms of inherited childhood-onset neurodegenerative disorders. They form a heterogeneous group of incurable lysosomal storage diseases that lead to blindness, motor deterioration, epilepsy, and dementia. Traditionally the NCL diseases were classified according to the age of disease onset (infantile, late-infantile, juvenile, and adult forms), with at least 13 different NCL varieties having been described at present. The current review focuses on classic juvenile NCL (JNCL) or the so-called Batten (Batten-Spielmeyer-Vogt; Spielmeyer-Sjogren) disease, which represents the most common and the most studied form of NCL, and is caused by mutations in the CLN3 gene located on human chromosome 16. Most JNCL patients carry the same 1.02-kb deletion in this gene, encoding an unusual transmembrane protein, CLN3, or battenin. Accordingly, the names CLN3-related neuronal ceroid lipofuscinosis or CLN3-disease sometimes have been used for this malady. Despite excessive in vitro and in vivo studies, the precise functions of the CLN3 protein and the JNCL disease mechanisms remain elusive and are the main subject of this review. Although the CLN3 gene is highly conserved in evolution of all mammalian species, detailed analysis of recent genomic and transcriptomic data indicates the presence of human-specific features of its expression, which are also under discussion. The main recorded to date changes in cell metabolism, to some extent contributing to the emergence and progression of JNCL disease, and human-specific molecular features of CLN3 gene expression are summarized and critically discussed with an emphasis on the possible molecular mechanisms of the malady appearance and progression.
    Keywords:  CLN3; CLN3 gene regulation; JNCL; biomarker POLR2J2; juvenile Batten disease; molecular mechanisms; neuronal ceroid lipofuscinoses
    DOI:  https://doi.org/10.3390/ijms21218055
  34. Aging (Albany NY). 2020 Nov 06. 12
    The therapeutic value of XL388 in human glioma cells.
    Shan Zhong, Jun Xue, Jiao-Jiao Cao, Bomin Sun, Qing-Fang Sun, Liu-Guan Bian, Liang-Yun Hu, Si-Jian Pan.
      XL388 is a highly efficient and orally-available ATP-competitive PI3K-mTOR dual inhibitor. Its activity against glioma cells was studied here. In established and primary human glioma cells, XL388 potently inhibited cell survival and proliferation as well as cell migration, invasion and cell cycle progression. The dual inhibitor induced significant apoptosis activation in glioma cells. In A172 cells and primary human glioma cells, XL388 inhibited Akt-mTORC1/2 activation by blocking phosphorylation of Akt and S6K1. XL388-induced glioma cell death was only partially attenuated by a constitutively-active mutant Akt1. Furthermore, it was cytotoxic against Akt1-knockout A172 glioma cells. XL388 downregulated MAF bZIP transcription factor G (MAFG) and inhibited Nrf2 signaling, causing oxidative injury in glioma cells. Conversely, antioxidants, n-acetylcysteine, pyrrolidine dithiocarbamate and AGI-106, alleviated XL388-induced cytotoxicity and apoptosis in glioma cells. Oral administration of XL388 inhibited subcutaneous A172 xenograft growth in severe combined immunodeficient mice. Akt-S6K1 inhibition and MAFG downregulation were detected in XL388-treated A172 xenograft tissues. Collectively, XL388 efficiently inhibits human glioma cell growth, through Akt-mTOR-dependent and -independent mechanisms.
    Keywords:  Akt; MAFG; XL388; glioma; mTOR
    DOI:  https://doi.org/10.18632/aging.103791
  35. Ageing Res Rev. 2020 Oct 29. pii: S1568-1637(20)30338-X. [Epub ahead of print] 101203
    The dialogue between the ubiquitin-proteasome system and autophagy: implications in ageing.
    Jia Liang Sun-Wang, Saška Ivanova, Antonio Zorzano.
      Dysregulated proteostasis is one of the hallmarks of ageing. Damaged proteins may impair cellular function and their accumulation may lead to tissue dysfunction and disease. This is why protective mechanisms to safeguard the cell proteome have evolved. These mechanisms consist of cellular machineries involved in protein quality control, including regulators of protein translation, folding, trafficking and degradation. In eukaryotic cells, protein degradation occurs via two main pathways: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. Although distinct pathways, they are not isolated systems and have a complementary nature, as evidenced by recent studies. These findings raise the question of how autophagy and the proteasome crosstalk. In this review we address how the two degradation pathways impact each other, thereby adding a new layer of regulation to protein degradation. We also analyze the implications of the UPS and autophagy in ageing.
    Keywords:  Ageing; UPS-autophagy crosstalk; autophagy; proteostasis; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.arr.2020.101203
  36. Bone. 2020 Oct 29. pii: S8756-3282(20)30517-2. [Epub ahead of print]143 115729
    Hip disease in Mucopolysaccharidoses and Mucolipidoses: A review of mechanisms, interventions and future perspectives.
    Esmee Oussoren, Margreet A E M Wagenmakers, Bianca Link, Jan C van der Meijden, W W M Pim Pijnappel, George J G Ruijter, Mirjam Langeveld, Ans T van der Ploeg.
      The hips are frequently involved in inheritable diseases which affect the bones. The clinical and radiological presentation of these diseases may be very similar to common hip disorders as developmental dysplasia of the hip, osteoarthritis and avascular necrosis, so the diagnosis may be easily overlooked and treatment may be suboptimal. Mucopolysaccharidosis (MPS) and Mucolipidosis (ML II and III) are lysosomal storage disorders with multisystemic involvement. Characteristic skeletal abnormalities, known as dysostosis multiplex, are common in MPS and ML and originate from intra-lysosomal storage of glycosaminoglycans in cells of the cartilage, bones and ligaments. The hip joint is severely affected in MPS and ML. Hip pathology results in limitations in mobility and pain from young age, and negatively affects quality of life. In order to better understand the underlying process that causes hip disease in MPS and ML, this review first describes the normal physiological (embryonic) hip joint development, including the interplay between the acetabulum and the femoral head. In the second part the factors contributing to altered hip morphology and function in MPS and ML are discussed, such as abnormal development of the pelvic- and femoral bones (which results in altered biomechanical forces) and inflammation. In the last part of this review therapeutic options and future perspectives are addressed.
    Keywords:  Acetabulum; Bone disease; Dysostosis multiplex; Embryonic development; Femoral head; Hip disease; Joint disease; ML; MPS; Mucolipidosis; Mucopolysaccharidosis; Osteoarthritis; Skeletal disease
    DOI:  https://doi.org/10.1016/j.bone.2020.115729
  37. FEBS J. 2020 Nov 03.
    Bafilomycin A1 enhances NLRP3 inflammasome activation in human monocytes independent of lysosomal acidification.
    Shi Yu, Jack Green, Rose Wellens, Gloria Lopez-Castejon, David Brough.
      The release of interleukin (IL)-1β from primary human monocytes in response to extracellular LPS occurs through the NLRP3 inflammasome. In primary monocytes, in response to LPS, NLRP3-inflammasome activation is characterised by an independence of K+ efflux and ASC speck formation, and has been termed the 'alternative' pathway. Here we report that pharmacological inhibition of V-ATPase with bafilomycin A1 exacerbated LPS-induced NLRP3 inflammasome activation in primary human monocytes. Inhibition of V-ATPase in the presence of extracellular LPS led to NLRP3-dependent, K+ efflux-independent, ASC oligomerization and caspase-1 activation. Although V-ATPases are required for lysosomal acidification, we found that acidic lysosomal pH and protease activity were dispensable for this altered response, suggesting that V-ATPase inhibition triggered alternative signalling events. Therefore, V-ATPases may serve additional roles during NLRP3 inflammasome activation in primary human monocytes.
    Keywords:  NLRP3; alternative NLRP3 inflammasome; caspase 1 (CASP1); cytokine; immune cell; immunology; inflammasome; inflammation; interleukin 1 (IL-1); monocyte
    DOI:  https://doi.org/10.1111/febs.15619
  38. Blood. 2020 Nov 04. pii: blood.2020007975. [Epub ahead of print]
    ER associated degradation preserves hematopoietic stem cell quiescence and self-renewal by restricting mTOR activity.
    Lu Liu, Ayaka Inoki, Kelly Fan, Fengbiao Mao, Guojun Shi, Xi Jin, Meiling Zhao, Gina Ney, Morgan Alexander Jones, Shengyi Sun, Yali Dou, Ken Inoki, Ling Qi, Qing Li.
      Hematopoietic stem cells (HSC) self-renew to sustain stem cell pools and differentiate to generate all types of blood cells. HSCs remain in quiescence to sustain their long-term self-renewal potential. It remains unclear whether protein quality control is required for stem cells in quiescence when RNA content, protein synthesis, and metabolic activities are profoundly reduced. Here, we report that protein quality control via endoplasmic reticulum associated degradation (ERAD) governs the function of quiescent hematopoietic stem cells (HSCs). The Sel1L/Hrd1 ERAD genes are enriched in the quiescent and inactive HSCs, and conditional knockout of Sel1L in hematopoietic tissues drives HSCs to hyper-proliferation, which leads to complete loss of HSC self-renewal and HSC depletion. Mechanistically, ERAD deficiency via Sel1L knockout leads to activation of mTOR signalling. Furthermore, we identify Rheb, an activator of mTOR, as a novel protein substrate of Sel1L/Hrd1 ERAD, which accumulates upon Sel1L deletion and HSC activation. Importantly, inhibition of mTOR, or Rheb, rescues HSC defects in Sel1L knockout mice. Protein quality control via ERAD is, therefore, a critical checkpoint that governs HSC quiescence and self-renewal by Rheb-mediated restriction of mTOR activity.
    DOI:  https://doi.org/10.1182/blood.2020007975
  39. Cell Death Dis. 2020 Nov 03. 11(11): 948
    Combined treatment of mitoxantrone sensitizes breast cancer cells to rapalogs through blocking eEF-2K-mediated activation of Akt and autophagy.
    Yidi Guan, Shilong Jiang, Wenling Ye, Xingcong Ren, Xinluan Wang, Yi Zhang, Mingzhu Yin, Kuansong Wang, Yongguang Tao, JinMing Yang, Dongsheng Cao, Yan Cheng.
      Oncogenic activation of the mTOR signaling pathway occurs frequently in tumor cells and contributes to the devastating features of cancer, including breast cancer. mTOR inhibitors rapalogs are promising anticancer agents in clinical trials; however, rapalogs resistance remains an unresolved clinical challenge. Therefore, understanding the mechanisms by which cells become resistant to rapalogs may guide the development of successful mTOR-targeted cancer therapy. In this study, we found that eEF-2K, which is overexpressed in cancer cells and is required for survival of stressed cells, was involved in the negative-feedback activation of Akt and cytoprotective autophagy induction in breast cancer cells in response to mTOR inhibitors. Therefore, disruption of eEF-2K simultaneously abrogates the two critical resistance signaling pathways, sensitizing breast cancer cells to rapalogs. Importantly, we identified mitoxantrone, an admitted anticancer drug for a wide range of tumors, as a potential inhibitor of eEF-2K via a structure-based virtual screening strategy. We further demonstrated that mitoxantrone binds to eEF-2K and inhibits its activity, and the combination treatment of mitoxantrone and mTOR inhibitor resulted in significant synergistic cytotoxicity in breast cancer. In conclusion, we report that eEF-2K contributes to the activation of resistance signaling pathways of mTOR inhibitor, suggesting a novel strategy to enhance mTOR-targeted cancer therapy through combining mitoxantrone, an eEF-2K inhibitor.
    DOI:  https://doi.org/10.1038/s41419-020-03153-x
  40. Cancer Discov. 2020 Nov 02.
    Targeting Metabolic Plasticity and Flexibility Dynamics for Cancer Therapy.
    Sarah-Maria Fendt, Christian Frezza, Ayelet Erez.
      Cancer cells continuously rewire their metabolism to fulfill their need for rapid growth and survival while subject to changes in environmental cues. Thus, a vital component of a cancer cell lies in its metabolic adaptability. The constant demand for metabolic alterations requires flexibility, that is, the ability to utilize different metabolic substrates; as well as plasticity, that is, the ability to process metabolic substrates in different ways. In this review, we discuss how dynamic changes in cancer metabolism affect tumor progression and the consequential implications for cancer therapy. SIGNIFICANCE: Recognizing cancer dynamic metabolic adaptability as an entity can lead to targeted therapy that is expected to decrease drug resistance.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-0844
  41. Sci Signal. 2020 Nov 03. pii: eaaz4003. [Epub ahead of print]13(656):
    Gβγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils.
    Natalie K Rynkiewicz, Karen E Anderson, Sabine Suire, Daniel M Collins, Eleftherios Karanasios, Oscar Vadas, Roger Williams, David Oxley, Jonathan Clark, Len R Stephens, Phillip T Hawkins.
      The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP3 production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to fMLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.
    DOI:  https://doi.org/10.1126/scisignal.aaz4003
  42. Metab Brain Dis. 2020 Nov 06.
    Fabry disease: GLA deletion alters a canonical splice site in a family with neuropsychiatric manifestations.
    Patrícia Varela, Gerson Carvalho, Renan Paulo Martin, João Bosco Pesquero.
      Fabry disease (FD) is a rare X-linked glycosphingolipidosis caused by mutations in GLA, a gene responsible for encoding α-galactosidase A, an enzyme required for degradation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in all cells of the body. FD patients present a broad spectrum of clinical phenotype and many symptoms are shared with other diseases, making diagnosis challenging. Here we describe a novel GLA variant located in the 5' splice site of the intron 3, in four members of a family with neuropsychiatric symptoms. Analysis of the RNA showed the variant promotes alteration of the wild type donor site, affecting splicing and producing two aberrant transcripts. The functional characterization showed absence of enzymatic activity in cells expressing both transcripts, confirming their pathogenicity. The family presents mild signs of FD, as angiokeratoma, cornea verticillata, acroparesthesia, tinnitus, vertigo, as well as accumulation of plasma lyso-Gb3 and urinary Gb3. Interestingly, the man and two women present psychiatric symptoms, as depression or schizophrenia. Although psychiatric illnesses, especially depression, are frequently reported in patients with FD and studies have shown that the hippocampus is an affected brain structure in these patients, it is not clear whether the Gb3 accumulation in the brain is responsible for these symptoms or they are secondary. Therefore, new studies are needed to understand whether the accumulation of Gb3 could produce neuronal alterations leading to psychiatric symptoms.
    Keywords:  Depression; Fabry disease; Neurological manifestations; Psychiatric symptoms, GLA gene; Schizophrenia; Splice site mutation; α-Galactosidase A
    DOI:  https://doi.org/10.1007/s11011-020-00640-0
  43. ACS Omega. 2020 Oct 27. 5(42): 26967-26977
    Fluorescent Probes for Super-Resolution Microscopy of Lysosomes.
    Aditya Yadav, Chethana Rao, Chayan K Nandi.
      Lysosomes are membrane-enclosed small spherical cytoplasmic organelles. Malfunctioning and abnormalities in lysosomes can cause a plethora of neurodegenerative diseases. Consequently, understanding the structural information on lysosomes down to a subnanometer level is essential. Recently, super-resolution imaging techniques enable us to visualize dynamical processes occurring in suborganelle structures inside living cells down to subnanometer accuracy by breaking the diffraction limit. A brighter and highly photostable fluorescent probe is essential for super-resolution microscopy. In this regard, this mini-review deals with the various types of super-resolution techniques and the probes that are used to specifically stain and resolve the structure of the lysosomes.
    DOI:  https://doi.org/10.1021/acsomega.0c04018
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