bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021–11–07
37 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates.
  2. Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation.
  3. Disruption of morphogenic and growth pathways in lysosomal storage diseases.
  4. TSC1 loss increases risk for tauopathy by inducing tau acetylation and preventing tau clearance via chaperone-mediated autophagy.
  5. Targeting eIF4F translation complex sensitizes B-ALL cells to tyrosine kinase inhibition.
  6. Regulation of mTORC1 by amino acids in mammalian cells: A general picture of recent advances.
  7. The Genetic Basis of Phenotypic Heterogeneity in the Neuronal Ceroid Lipofuscinoses.
  8. Exploiting lysosomal advantage in tumors.
  9. Loss of Hepatic Transcription Factor EB Attenuates Alcohol-Associated Liver Carcinogenesis.
  10. A gene toolbox for monitoring autophagy transcription.
  11. Hayatine inhibits amino acid-induced mTORC1 activation as a novel mTOR-Rag A/C interaction disruptor.
  12. Activation of Akt-mTORC1 signalling reverts cancer-dependent muscle wasting.
  13. GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.
  14. Plasmacytoid dendritic cell activation is dependent on coordinated expression of distinct amino acid transporters.
  15. Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218.
  16. Ajugol enhances TFEB-mediated lysosome biogenesis and lipophagy to alleviate non-alcoholic fatty liver disease.
  17. Backbone 1H, 15N, and 13C resonance assignments of the Phafin2 pleckstrin homology domain.
  18. Two Distinct Lysosomal Targeting Strategies Afford Trojan Horse Antibodies With Pan-Filovirus Activity.
  19. Amino Acid Homeostasis in Mammalian Cells with a Focus on Amino Acid Transport.
  20. Cryo-EM structures and transport mechanism of human P5B type ATPase ATP13A2.
  21. Loss of tyrosine catabolic enzyme HPD promotes glutamine anaplerosis through mTOR signaling in liver cancer.
  22. Exosome Biogenesis and Lysosome Function Determine Podocyte Exosome Release and Glomerular Inflammatory Response during Hyperhomocysteinemia.
  23. Fibroblast Growth Factor 23 (FGF23) leads to endolysosomal routing of the renal phosphate co-transporters NaPi-IIa and NaPi-IIc in vivo.
  24. Involvement of the Choroid Plexus in the Pathogenesis of Niemann-Pick Disease Type C.
  25. Phagosome maturation in macrophages: Eat, digest, adapt, and repeat.
  26. Early Endosomal GTPase Rab5 Regulates GPIb Trafficking and Platelet Production In Vitro.
  27. Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.
  28. Cryo-EM structures of PI3Kα reveal conformational changes during inhibition and activation.
  29. The EMT activator ZEB1 accelerates endosomal trafficking to establish a polarity axis in lung adenocarcinoma cells.
  30. The involvement of sodium in the function of the human amino acid transporter ASCT2.
  31. The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7.
  32. Clinical features of Danon disease and insights gained from LAMP-2 deficiency models.
  33. FGFR-inhibitor-mediated dismissal of SWI/SNF complexes from YAP-dependent enhancers induces adaptive therapeutic resistance.
  34. SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase.
  35. Lipid-induced DRAM recruits STOM to lysosomes and induces LMP to promote exosome release from hepatocytes in NAFLD.
  36. Long noncoding RNA KB-1460A1.5 inhibits glioma tumorigenesis via miR-130a-3p/TSC1/mTOR/YY1 feedback loop.
  37. Evaluation of the long-term treatment effects of intravenous idursulfase in patients with mucopolysaccharidosis II (MPS II) using statistical modeling: data from the Hunter Outcome Survey (HOS).
  1. Nat Commun. 2021 Nov 04. 12(1): 6393
    Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates.
    Helena Costa-Verdera, Fanny Collaud, Christopher R Riling, Pauline Sellier, Jayme M L Nordin, G Michael Preston, Umut Cagin, Julien Fabregue, Simon Barral, Maryse Moya-Nilges, Jacomina Krijnse-Locker, Laetitia van Wittenberghe, Natalie Daniele, Bernard Gjata, Jeremie Cosette, Catalina Abad, Marcelo Simon-Sola, Severine Charles, Mathew Li, Marco Crosariol, Tom Antrilli, William J Quinn, David A Gross, Olivier Boyer, Xavier M Anguela, Sean M Armour, Pasqualina Colella, Giuseppe Ronzitti, Federico Mingozzi.
      Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.
    DOI:  https://doi.org/10.1038/s41467-021-26744-4
  2. Nat Commun. 2021 Nov 04. 12(1): 6409
    Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation.
    Chih-Yao Chung, Kritarth Singh, Vassilios N Kotiadis, Gabriel E Valdebenito, Jee Hwan Ahn, Emilie Topley, Joycelyn Tan, William D Andrews, Benoit Bilanges, Robert D S Pitceathly, Gyorgy Szabadkai, Mariia Yuneva, Michael R Duchen.
      Mutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy - tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient-derived cells carrying the most prevalent disease related mtDNA mutation, the m.3243 A > G. These studies reveal that the mutation promotes changes in metabolites which are associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 reduced mtDNA mutant load and partially rescued cellular bioenergetic function. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243 A > G mutation.
    DOI:  https://doi.org/10.1038/s41467-021-26746-2
  3. WIREs Mech Dis. 2021 Sep;13(5): e1521
    Disruption of morphogenic and growth pathways in lysosomal storage diseases.
    Thiago Corrêa, Bruno C Feltes, Roberto Giugliani, Ursula Matte.
      The lysosome achieved a new protagonism that highlights its multiple cellular functions, such as in the catabolism of complex substrates, nutrient sensing, and signaling pathways implicated in cell metabolism and growth. Lysosomal storage diseases (LSDs) cause lysosomal accumulation of substrates and deficiency in trafficking of macromolecules. The substrate accumulation can impact one or several pathways which contribute to cell damage. Autophagy impairment and immune response are widely studied, but less attention is paid to morphogenic and growth pathways and its impact on the pathophysiology of LSDs. Hedgehog pathway is affected with abnormal expression and changes in distribution of protein levels, and a reduced number and length of primary cilia. Moreover, growth pathways are identified with delay in reactivation of mTOR that deregulate termination of autophagy and reformation of lysosomes. Insulin resistance caused by changes in lipids rafts has been described in different LSDs. While the genetic and biochemical bases of deficient proteins in LSDs are well understood, the secondary molecular mechanisms that disrupt wider biological processes associated with LSDs are only now becoming clearer. Therefore, we explored how specific signaling pathways can be related to specific LSDs, showing that a system medicine approach could be a valuable tool for the better understanding of LSD pathogenesis. This article is categorized under: Metabolic Diseases > Molecular and Cellular Physiology.
    Keywords:  Hh pathway; LSDs; insulin; mTOR; systems medicine
    DOI:  https://doi.org/10.1002/wsbm.1521
  4. Sci Adv. 2021 Nov 05. 7(45): eabg3897
    TSC1 loss increases risk for tauopathy by inducing tau acetylation and preventing tau clearance via chaperone-mediated autophagy.
    Carolina Alquezar, Kathleen M Schoch, Ethan G Geier, Eliana Marisa Ramos, Aurora Scrivo, Kathy H Li, Andrea R Argouarch, Elisabeth E Mlynarski, Beth Dombroski, Michael DeTure, Dennis W Dickson, Jennifer S Yokoyama, Ana M Cuervo, Alma L Burlingame, Gerard D Schellenberg, Timothy M Miller, Bruce L Miller, Aimee W Kao.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/sciadv.abg3897
  5. Sci Rep. 2021 Nov 04. 11(1): 21689
    Targeting eIF4F translation complex sensitizes B-ALL cells to tyrosine kinase inhibition.
    Thanh-Trang Vo, Lee-Or Herzog, Roberta Buono, Jong-Hoon Scott Lee, Sharmila Mallya, Madeleine R Duong, Joshua Thao, Moran Gotesman, David A Fruman.
      The mechanistic target of rapamycin (mTOR) is a kinase whose activation is associated with poor prognosis in pre-B cell acute lymphoblastic leukemia (B-ALL). These and other findings have prompted diverse strategies for targeting mTOR signaling in B-ALL and other B-cell malignancies. In cellular models of Philadelphia Chromosome-positive (Ph+) B-ALL, mTOR kinase inhibitors (TOR-KIs) that inhibit both mTOR-complex-1 (mTORC1) and mTOR-complex-2 (mTORC2) enhance the cytotoxicity of tyrosine kinase inhibitors (TKIs) such as dasatinib. However, TOR-KIs have not shown substantial efficacy at tolerated doses in blood cancer clinical trials. Selective inhibition of mTORC1 or downstream effectors provides alternative strategies that may improve selectivity towards leukemia cells. Of particular interest is the eukaryotic initiation factor 4F (eIF4F) complex that mediates cap-dependent translation. Here we use novel chemical and genetic approaches to show that selective targeting of either mTORC1 kinase activity or components of the eIF4F complex sensitizes murine BCR-ABL-dependent pre-B leukemia cells to dasatinib. SBI-756, a small molecule inhibitor of eIF4F assembly, sensitizes human Ph+ and Ph-like B-ALL cells to dasatinib cytotoxicity without affecting survival of T lymphocytes or natural killer cells. These findings support the further evaluation of eIF4F-targeted molecules in combination therapies with TKIs in B-ALL and other blood cancers.
    DOI:  https://doi.org/10.1038/s41598-021-00950-y
  6. Anim Nutr. 2021 Dec;7(4): 1009-1023
    Regulation of mTORC1 by amino acids in mammalian cells: A general picture of recent advances.
    Shizhe Zhang, Xueyan Lin, Qiuling Hou, Zhiyong Hu, Yun Wang, Zhonghua Wang.
      The mechanistic target of rapamycin complex 1 (mTORC1) integrates various types of signal inputs, such as energy, growth factors, and amino acids to regulate cell growth and proliferation mainly through the 2 direct downstream targets, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and ribosomal protein S6 kinase 1 (S6K1). Most of the signal arms upstream of mTORC1 including energy status, stress signals, and growth factors converge on the tuberous sclerosis complex (TSC) - Ras homologue enriched in brain (Rheb) axis. Amino acids, however, are distinct from other signals and modulate mTORC1 using a unique pathway. In recent years, the transmission mechanism of amino acid signals upstream of mTORC1 has been gradually elucidated, and some sensors or signal transmission pathways for individual amino acids have also been discovered. With the help of these findings, we propose a general picture of recent advances, which demonstrates that various amino acids from lysosomes, cytoplasm, and Golgi are sensed by their respective sensors. These signals converge on mTORC1 and form a huge and complicated signal network with multiple synergies, antagonisms, and feedback mechanisms.
    Keywords:  Amino acids; Leucine; Mammal; Signaling pathway; mTORC1
    DOI:  https://doi.org/10.1016/j.aninu.2021.05.003
  7. Front Neurol. 2021 ;12 754045
    The Genetic Basis of Phenotypic Heterogeneity in the Neuronal Ceroid Lipofuscinoses.
    Emily Gardner, Sara E Mole.
      The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders that affect children and adults. They share some similar clinical features and the accumulation of autofluorescent storage material. Since the discovery of the first causative genes, more than 530 mutations have been identified across 13 genes in cases diagnosed with NCL. These genes encode a variety of proteins whose functions have not been fully defined; most are lysosomal enzymes, or transmembrane proteins of the lysosome or other organelles. Many mutations in these genes are associated with a typical NCL disease phenotype. However, increasing numbers of variant disease phenotypes are being described, affecting age of onset, severity or progression, and including some distinct clinical phenotypes. This data is collated by the NCL Mutation Database which allows analysis from many perspectives. This article will summarise and interpret current knowledge and understanding of their genetic basis and phenotypic heterogeneity.
    Keywords:  CLN; NCL; batten disease; gene; lysosomal disease; mutation; neuronal ceroid lipofuscinosis
    DOI:  https://doi.org/10.3389/fneur.2021.754045
  8. Sci Signal. 2021 Nov 02. 14(707): eabn0232
    Exploiting lysosomal advantage in tumors.
    Leslie K Ferrarelli.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/scisignal.abn0232
  9. Am J Pathol. 2021 Oct 27. pii: S0002-9440(21)00442-9. [Epub ahead of print]
    Loss of Hepatic Transcription Factor EB Attenuates Alcohol-Associated Liver Carcinogenesis.
    Xiaojuan Chao, Shaogui Wang, Madeline Hlobik, Andrea Ballabio, Hong-Min Ni, Wen-Xing Ding.
      Alcohol is a well-known risk factor for hepatocellular carcinoma (HCC). Autophagy plays a dual role in liver cancer, as it suppresses tumor initiation and promotes tumor progression. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy, which is impaired in alcohol-related liver disease. However, the role of TFEB in alcohol-associated liver carcinogenesis is unknown. Liver-specific Tfeb knockout (KO) mice and their matched wild type (WT) littermates were injected with the carcinogen diethylnitrosamine (DEN), followed by chronic ethanol feeding. We found that the numbers of both total and larger tumors increased significantly in DEN-treated mice fed with ethanol diet than in mice fed with control diet. While the number of tumors was not different between WT and L-Tfeb KO mice fed either control or ethanol diet, the number of larger tumors was less in L-Tfeb KO mice than in WT mice. To our surprise, no differences were found in liver injury, steatosis, inflammation, ductular reaction, fibrosis and tumor cell proliferation in DEN-treated mice fed ethanol. However, the levels of glypican 3, a marker of malignant HCC, markedly decreased in DEN-treated L-Tfeb KO mice fed ethanol in comparison to the WT mice. Our findings indicate that chronic ethanol feeding promotes DEN-initiated liver tumor development, which tends to be attenuated by genetic deletion of hepatic TFEB.
    Keywords:  Autophagy; HCC; Lysosome; NAFLD; Steatosis
    DOI:  https://doi.org/10.1016/j.ajpath.2021.10.004
  10. Cell Death Dis. 2021 Nov 02. 12(11): 1044
    A gene toolbox for monitoring autophagy transcription.
    Matteo Bordi, Rossella De Cegli, Beatrice Testa, Ralph A Nixon, Andrea Ballabio, Francesco Cecconi.
      Autophagy is a highly dynamic and multi-step process, regulated by many functional protein units. Here, we have built up a comprehensive and up-to-date annotated gene list for the autophagy pathway, by combining previously published gene lists and the most recent publications in the field. We identified 604 genes and created main categories: MTOR and upstream pathways, autophagy core, autophagy transcription factors, mitophagy, docking and fusion, lysosome and lysosome-related genes. We then classified such genes in sub-groups, based on their functions or on their sub-cellular localization. Moreover, we have curated two shorter sub-lists to predict the extent of autophagy activation and/or lysosomal biogenesis; we next validated the "induction list" by Real-time PCR in cell lines during fasting or MTOR inhibition, identifying ATG14, ATG7, NBR1, ULK1, ULK2, and WDR45, as minimal transcriptional targets. We also demonstrated that our list of autophagy genes can be particularly useful during an effective RNA-sequencing analysis. Thus, we propose our lists as a useful toolbox for performing an informative and functionally-prognostic gene scan of autophagy steps.
    DOI:  https://doi.org/10.1038/s41419-021-04121-9
  11. Biochem Biophys Res Commun. 2021 Oct 21. pii: S0006-291X(21)01412-1. [Epub ahead of print]583 71-78
    Hayatine inhibits amino acid-induced mTORC1 activation as a novel mTOR-Rag A/C interaction disruptor.
    Meiling Lu, Lei Yu, Yanrong Yang, Jiali Zhu, Sujing Qiang, Xinbo Wang, Jia Wang, Xiao Tan, Weifeng Wang, Yue Zhang, Weichao Wang, Jian Xie, Xinyan Chen, Hongbing Wang, Xianghuan Cui, Xin Ge.
      Abnormal activation of the mechanistic target of rapamycin (mTOR) signaling is commonly observed in many cancers and attracts extensive attention as an oncology drug discovery target, which is encouraged by the success of rapamycin and its analogs (rapalogs) in treatment of mTORC1-hyperactive cancers in both pre-clinic models and clinical trials. However, rapamycin and existing rapalogs have typically short-lasting partial responses due to drug resistance, thereby triggering our interest to investigate a potential mTORC1 inhibitor that is mechanistically different from rapamycin. Here, we report that hayatine, a derivative from Cissampelos, can serve as a potential mTORC1 inhibitor selected from a natural compound library. The unique properties owned by hayatine such as downregulation of mTORC1 activities, induction of mTORC1's translocation to lysosomes followed by autophagy, and suppression on cancer cell growth, strongly emphasize its role as a potential mTORC1 inhibitor. Mechanistically, we found that hayatine disrupts the interaction between mTORC1 complex and its lysosomal adaptor RagA/C by binding to the hydrophobic loop of RagC, leading to mTORC1 inhibition that holds great promise to overcome rapamycin resistance. Taken together, our data shed light on an innovative strategy using structural interruption-based mTORC1 inhibitors for cancer treatment.
    Keywords:  Hayatine; Rag A/C; Rapamycin; mTOR
    DOI:  https://doi.org/10.1016/j.bbrc.2021.10.014
  12. J Cachexia Sarcopenia Muscle. 2021 Nov 06.
    Activation of Akt-mTORC1 signalling reverts cancer-dependent muscle wasting.
    Alessia Geremia, Roberta Sartori, Martina Baraldo, Leonardo Nogara, Valeria Balmaceda, Georgia Ana Dumitras, Stefano Ciciliot, Marco Scalabrin, Hendrik Nolte, Bert Blaauw.
       BACKGROUND: Cancer-related muscle wasting occurs in most cancer patients. An important regulator of adult muscle mass and function is the Akt-mTORC1 pathway. While Akt-mTORC1 signalling is important for adult muscle homeostasis, it is also a major target of numerous cancer treatments. Which role Akt-mTORC1 signalling plays during cancer cachexia in muscle is currently not known. Here, we aimed to determine how activation or inactivation of the pathway affects skeletal muscle during cancer cachexia.
    METHODS: We used inducible, muscle-specific Raptor ko (mTORC1) mice to determine the effect of reduced mTOR signalling during cancer cachexia. On the contrary, in order to understand if skeletal muscles maintain their anabolic capacity and if activation of Akt-mTORC1 signalling can reverse cancer cachexia, we generated mice in which we can inducibly activate Akt specifically in skeletal muscles.
    RESULTS: We found that mTORC1 signalling is impaired during cancer cachexia, using the Lewis lung carcinoma and C26 colon cancer model, and is accompanied by a reduction in protein synthesis rates of 57% (P < 0.01). Further reduction of mTOR signalling, as seen in Raptor ko animals, leads to a 1.5-fold increase in autophagic flux (P > 0.001), but does not further increase muscle wasting. On the other hand, activation of Akt-mTORC1 signalling in already cachectic animals completely reverses the 15-20% loss in muscle mass and force (P < 0.001). Interestingly, Akt activation only in skeletal muscle completely normalizes the transcriptional deregulation observed in cachectic muscle, despite having no effect on tumour size or spleen mass. In addition to stimulating muscle growth, it is also sufficient to prevent the increase in protein degradation normally observed in muscles from tumour-bearing animals.
    CONCLUSIONS: Here, we show that activation of Akt-mTORC1 signalling is sufficient to completely revert cancer-dependent muscle wasting. Intriguingly, these results show that skeletal muscle maintains its anabolic capacities also during cancer cachexia, possibly giving a rationale behind some of the beneficial effects observed in exercise in cancer patients.
    Keywords:  Akt; Cancer cachexia; Muscle growth; Raptor; Skeletal muscle force; mTOR
    DOI:  https://doi.org/10.1002/jcsm.12854
  13. Autophagy. 2021 Nov 05. 1-17
    GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.
    Sonia Brun, Eloïne Bestion, Eric Raymond, Firas Bassissi, Zuzana Macek Jilkova, Soraya Mezouar, Madani Rachid, Marie Novello, Jennifer Tracz, Ahmed Hamaï, Gilles Lalmanach, Lise Vanderlynden, Raphael Legouffe, Jonathan Stauber, Thomas Schubert, Maximilian G Plach, Jérôme Courcambeck, Cyrille Drouot, Guillaume Jacquemot, Cindy Serdjebi, Gael Roth, Jean-Pierre Baudoin, Christelle Ansaldi, Thomas Decaens, Philippe Halfon.
      Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment.Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC50: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH4Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn2+: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone.
    Keywords:  Antitumor; PPT1; autophagy; liver cancer; lysosome; mtor
    DOI:  https://doi.org/10.1080/15548627.2021.1988357
  14. Immunity. 2021 Oct 23. pii: S1074-7613(21)00446-5. [Epub ahead of print]
    Plasmacytoid dendritic cell activation is dependent on coordinated expression of distinct amino acid transporters.
    Katarzyna M Grzes, David E Sanin, Agnieszka M Kabat, Michal A Stanczak, Joy Edwards-Hicks, Mai Matsushita, Alexandra Hackl, Fabian Hässler, Kristin Knoke, Sophie Zahalka, Matteo Villa, David M Kofler, Reinhard E Voll, Paola Zigrino, Mario Fabri, Erika L Pearce, Edward J Pearce.
      Human plasmacytoid dendritic cells (pDCs) are interleukin-3 (IL-3)-dependent cells implicated in autoimmunity, but the role of IL-3 in pDC biology is poorly understood. We found that IL-3-induced Janus kinase 2-dependent expression of SLC7A5 and SLC3A2, which comprise the large neutral amino acid transporter, was required for mammalian target of rapamycin complex 1 (mTORC1) nutrient sensor activation in response to toll-like receptor agonists. mTORC1 facilitated increased anabolic activity resulting in type I interferon, tumor necrosis factor, and chemokine production and the expression of the cystine transporter SLC7A11. Loss of function of these amino acid transporters synergistically blocked cytokine production by pDCs. Comparison of in vitro-activated pDCs with those from lupus nephritis lesions identified not only SLC7A5, SLC3A2, and SLC7A11 but also ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) as components of a shared transcriptional signature, and ENPP2 inhibition also blocked cytokine production. Our data identify additional therapeutic targets for autoimmune diseases in which pDCs are implicated.
    Keywords:  GM-CSF; IL-3; JAK-STAT signaling; amino acid transporters; autoimmunity; cytokines; mTORC1; metabolism; plasmacytoid dendritic cells
    DOI:  https://doi.org/10.1016/j.immuni.2021.10.009
  15. Acta Pharm Sin B. 2021 Oct;11(10): 3178-3192
    Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218.
    Po Hu, Hui Li, Wenzhuo Sun, Hongzheng Wang, Xiaoxuan Yu, Yingjie Qing, Zhanyu Wang, Mengyuan Zhu, Jingyan Xu, Qinglong Guo, Hui Hui.
      The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth in vivo. Thus, the necessary impact of integral lysosomal function in cell rescue and death were illustrated.
    Keywords:  AO, acridine orange; ATG, autophagy related; BAF A1, bafilomycin A1; BID, BH3-interacting domain death agonist; CCK8, Cell Counting Kit; CTSB, cathepsin B; CTSD, cathepsin D; CaN, calcineurin; Cathepsin D; Cholesterol; CsA, cyclosporine A; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DCFH-DA, 2,7-dichlorodi-hydrofluorescein diacetate; Dex, dexamethasone; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; FBS, fetal bovine serum; Hematological malignancies; K48, lysine 48; K63, lysine 63; LAMPs, lysosomal-associated membrane proteins; LC3, microtubule-associated protein 1 light chain 3; LCD, lysosome-dependent cell death; LMP, lysosome membrane permeabilization; LW-218; Lysophagy; Lysosomal damage; Lysosomal membrane permeabilization; Lysosome-dependent cell death; NH4Cl, ammonium chloride; NPC, Niemann-Pick type disease C; NPC1, NPC intracellular cholesterol transporter 1; OD, optical density; P62/SQSTM1, sequestosome 1; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; RAB7A, RAS-related protein RAB-7a; ROS, reactive oxygen species; RT-qPCR, real time quantitative PCR; TFEB, transcription factor EB; TRPML1, transient receptor potential mucolipin 1; shRNA, short hairpin RNA
    DOI:  https://doi.org/10.1016/j.apsb.2021.02.004
  16. Pharmacol Res. 2021 Oct 31. pii: S1043-6618(21)00548-X. [Epub ahead of print] 105964
    Ajugol enhances TFEB-mediated lysosome biogenesis and lipophagy to alleviate non-alcoholic fatty liver disease.
    Heng Zhang, Junfeng Lu, Hao Liu, Lingling Guan, Shiqing Xu, Zai Wang, Yang Qiu, Honglin Liu, Liang Peng, Xiuli Men.
      Lipophagy is the autophagic degradation of lipid droplets. Dysregulated lipophagy has been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Ajugol is an active alkaloid isolated from the root of Rehmannia glutinosa which is commonly used to treat various inflammatory and metabolic diseases. This study aimed to investigate the effect of ajugol on alleviating hepatic steatosis and sought to determine whether its potential mechanism via the key lysosome-mediated process of lipophagy. Our findings showed that ajugol significantly improved high-fat diet-induced hepatic steatosis in mice and inhibited palmitate-induced lipid accumulation in hepatocytes. Further analysis found that hepatic steatosis promoted the expression of LC3-II, an autophagosome marker, but led to autophagic flux blockade due to a lack of lysosomes. Ajugol also enhanced lysosomal biogenesis and promoted the fusion of autophagosome and lysosome to improve impaired autophagic flux and hepatosteatosis. Mechanistically, ajugol inactivated mammalian target of rapamycin and induced nuclear translocation of the transcription factor EB (TFEB), an essential regulator of lysosomal biogenesis. siRNA-mediated knockdown of TFEB significantly abrogated ajugol-induced lysosomal biogenesis as well as autophagosome-lysosome fusion and lipophagy. We conclude that lysosomal deficit is a critical mediator of hepatic steatosis, and ajugol may alleviate NAFLD via promoting the TFEB-mediated autophagy-lysosomal pathway and lipophagy.
    Keywords:  Ajugol; Autophagic flux; BODIPY 493/503 (PubChem: CID: 134716599); Carboxymethylcellulose (CMC-Na) (PubChem CID: 24748); DMSO (PubChem CID: 679); Lipophagy; NAFLD; PVDF (PubChem CID: 3082294); Phenylmethylsulfonyl fluoride (PMSF) (PubChem CID: 4784); TFEB
    DOI:  https://doi.org/10.1016/j.phrs.2021.105964
  17. Biomol NMR Assign. 2021 Nov 05.
    Backbone 1H, 15N, and 13C resonance assignments of the Phafin2 pleckstrin homology domain.
    Jeffrey F Ellena, Tuo-Xian Tang, Narasimhamurthy Shanaiah, Daniel G S Capelluto.
      Phafin2 is a peripheral protein that triggers cellular signaling from endosomal and lysosomal compartments. The specific subcellular localization of Phafin2 is mediated by the presence of a tandem of phosphatidylinositol 3-phosphate (PtdIns3P)-binding domains, the pleckstrin homology (PH) and the Fab-1, YOTB, Vac1, and EEA1 (FYVE) domains. The requirement for both domains for binding to PtdIns3P still remains unclear. To understand the molecular interactions of the Phafin2 PH domain in detail, we report its nearly complete 1H, 15N, and 13C backbone resonance assignments.
    Keywords:  Endosome; FYVE domain; PH domain; Phafin2; Phosphoinositide
    DOI:  https://doi.org/10.1007/s12104-021-10054-3
  18. Front Immunol. 2021 ;12 729851
    Two Distinct Lysosomal Targeting Strategies Afford Trojan Horse Antibodies With Pan-Filovirus Activity.
    Ariel S Wirchnianski, Anna Z Wec, Elisabeth K Nyakatura, Andrew S Herbert, Megan M Slough, Ana I Kuehne, Eva Mittler, Rohit K Jangra, Jonathan Teruya, John M Dye, Jonathan R Lai, Kartik Chandran.
      Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability-the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a 'Trojan horse' therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses.
    Keywords:  Ebola; IGF2; Marburg; NPC1; NPC2; Trojan Horse bispecific antibodies; cryptic epitopes ; filovirus
    DOI:  https://doi.org/10.3389/fimmu.2021.729851
  19. J Nutr. 2021 Oct 27. pii: nxab342. [Epub ahead of print]
    Amino Acid Homeostasis in Mammalian Cells with a Focus on Amino Acid Transport.
    Stefan Bröer, Gregory Gauthier-Coles.
      Amino acid homeostasis is maintained by import, export, oxidation, and synthesis of nonessential amino acids, and by the synthesis and breakdown of protein. These processes work in conjunction with regulatory elements that sense amino acids or their metabolites. During and after nutrient intake, amino acid homeostasis is dominated by autoregulatory processes such as transport and oxidation of excess amino acids. Amino acid deprivation triggers processes such as autophagy and the execution of broader transcriptional programs to maintain plasma amino acid concentrations. Amino acid transport plays a crucial role in the absorption of amino acids in the intestine, the distribution of amino acids across cells and organs, the recycling of amino acids in the kidney, and the recycling of amino acids after protein breakdown.
    Keywords:  ATF4; GCN2; autophagy; mTORC1; solute carrier; transceptor
    DOI:  https://doi.org/10.1093/jn/nxab342
  20. Cell Discov. 2021 Nov 02. 7(1): 106
    Cryo-EM structures and transport mechanism of human P5B type ATPase ATP13A2.
    Xudong Chen, Mingze Zhou, Sensen Zhang, Jian Yin, Ping Zhang, Xujun Xuan, Peiyi Wang, Zhiqiang Liu, Boda Zhou, Maojun Yang.
      Polyamines are important polycations that play critical roles in mammalian cells. ATP13A2 belongs to the orphan P5B adenosine triphosphatases (ATPase) family and has been established as a lysosomal polyamine exporter to maintain the normal function of lysosomes and mitochondria. Previous studies have reported that several human neurodegenerative disorders are related to mutations in the ATP13A2 gene. However, the transport mechanism of ATP13A2 in the lysosome remains unclear. Here, we report the cryo-electron microscopy (cryo-EM) structures of three distinct intermediates of the human ATP13A2, revealing key insights into the spermine (SPM) transport cycle in the lysosome. The transmembrane domain serves as a substrate binding site and the C-terminal domain is essential for protein stability and may play a regulatory role. These findings advance our understanding of the polyamine transport mechanism, the lipid-associated regulation, and the disease-associated mutants of ATP13A2.
    DOI:  https://doi.org/10.1038/s41421-021-00334-6
  21. Cell Rep. 2021 Nov 02. pii: S2211-1247(21)01455-8. [Epub ahead of print]37(5): 109976
    Loss of tyrosine catabolic enzyme HPD promotes glutamine anaplerosis through mTOR signaling in liver cancer.
    Man Tong, Tin-Lok Wong, Hongzhi Zhao, Yuanyuan Zheng, Yu-Nong Xie, Cheuk-Hin Li, Lei Zhou, Noélia Che, Jing-Ping Yun, Kwan Man, Terence Kin-Wah Lee, Zongwei Cai, Stephanie Ma.
      
    DOI:  https://doi.org/10.1016/j.celrep.2021.109976
  22. Am J Pathol. 2021 Oct 27. pii: S0002-9440(21)00443-0. [Epub ahead of print]
    Exosome Biogenesis and Lysosome Function Determine Podocyte Exosome Release and Glomerular Inflammatory Response during Hyperhomocysteinemia.
    Dandan Huang, Guangbi Li, Owais M Bhat, Yao Zou, Ningjun Li, Joseph K Ritter, Pin-Lan Li.
      NLRP3 inflammasome activation in podocytes has been reported to be associated with enhanced release of exosomes containing NLRP3 inflammasome products from these cells during hyperhomocysteinemia (hHcy). In the present study, we examined the possible role of increased exosome secretion during podocyte NLRP3 inflammasome activation in the glomerular inflammatory response and tested whether exosome biogenesis and lysosome function are involved in the regulation of exosome release from podocytes during hHcy in mice and upon stimulation of homocysteine (Hcy) in podocytes. By nanoparticle tracking analysis, treatments of mice with amitriptyline (acid sphingomyelinase inhibitor), GW4869 (exosome biogenesis inhibitor), and rapamycin (lysosome function enhancer) were found to inhibit the elevation of urinary exosomes during hHcy. By examination of NLRP3 inflammasome activation in glomeruli during hHcy, only amitriptyline, but not GW4869 and rapamycin was shown to have inhibitory effect. However, all treatments attenuated glomerular inflammation and injury during hHcy. In cell studies, we demonstrated that Hcy treatment stimulated exosome release from podocytes, which was prevented by amitriptyline, GW4869, and rapamycin. Structured illumination microscopy showed that Hcy inhibited lysosome-MVB interactions in podocytes, which was prevented by amitriptyline or rapamycin, but not GW4869. It is concluded that activation of exosome biogenesis and dysregulated lysosome function are critically implicated in the enhancement of exosome release from podocytes leading to glomerular inflammation and injury during hHcy.
    DOI:  https://doi.org/10.1016/j.ajpath.2021.10.005
  23. Am J Physiol Renal Physiol. 2021 Nov 01.
    Fibroblast Growth Factor 23 (FGF23) leads to endolysosomal routing of the renal phosphate co-transporters NaPi-IIa and NaPi-IIc in vivo.
    Catharina J Küng, Betül Haykir, Udo Schnitzbauer, Daniela Egli-Spichtig, Nati Hernando, Carsten A Wagner.
      The sodium-dependentphosphate co-transporters NaPi-IIa and NaPi-IIc located at the brush border membrane of renal proximal tubules are regulated by numerous factors, including fibroblast growth factor 23 (FGF23). FGF23 downregulates NaPi-IIa and NaPi-IIc abundance after activating a signaling pathway involving phosphorylation of the extracellular signal-regulated protein kinase (phospho-ERK1/2). FGF23 also downregulates the expression of renal 1-α-hydroxylase (Cyp27b1) and upregulates 24-hydroxylase (Cyp24a1), thus reducing plasma calcitriol levels. Here, we examined the time course of the FGF23-induced internalization of NaPi-IIa and NaPi-IIc and their intracellular pathway towards degradation in vivo. Mice were injected intraperitoneally with recombinant human FGF23 (rh-FGF23) in the absence (biochemical analysis) or presence (immunohistochemistry) of leupeptin, an inhibitor of lysosomal proteases. Phosphorylation of ERK1/2 was enhanced 60 minutes after rh-FGF23 administration, and increased phosphorylation was still detected 480 minutes post-injection. Co-localization of phospho-ERK1/2 with NaPi-IIa was seen at 60, 120 and partly at 480 minutes. The abundance of both co-transporters was reduced 240 minutes after rh-FGF23 administration, with a further reduction at 480 minutes. NaPi-IIa and NaPi-IIc were found to co-localize with clathrin and early endosomal antigen 1 (EEA1) as early as 120 minutes after rh-FGF23 injection. Both co-transporters partially co-localized with cathepsin B and Lamp1, markers of lysosomes, 120 minutes after rh-FGF23 injection. Thus, NaPi-IIa and NaPi-IIc are internalized within 2 hours upon rh-FGF23 injection. Both co-transporters share the pathway of clathrin-mediated endocytosis that leads first to early endosomes, finally resulting in trafficking towards the lysosome as early as 120 minutes after rh-FGF23 administration.
    Keywords:  FGF23; Phosphate; calcitriol; endocytosis; proximal tubule
    DOI:  https://doi.org/10.1152/ajprenal.00250.2021
  24. Front Cell Neurosci. 2021 ;15 757482
    Involvement of the Choroid Plexus in the Pathogenesis of Niemann-Pick Disease Type C.
    Lien Van Hoecke, Caroline Van Cauwenberghe, Kristina Dominko, Griet Van Imschoot, Elien Van Wonterghem, Jonas Castelein, Junhua Xie, Wouter Claeys, Charysse Vandendriessche, Anna Kremer, Peter Borghgraef, Riet De Rycke, Silva Hecimovic, Roosmarijn E Vandenbroucke.
      Niemann-Pick type C (NPC) disease, sometimes called childhood Alzheimer's, is a rare neurovisceral lipid storage disease with progressive neurodegeneration leading to premature death. The disease is caused by loss-of-function mutations in the Npc1 or Npc2 gene which both result into lipid accumulation in the late endosomes and lysosomes. Since the disease presents with a broad heterogenous clinical spectrum, the involved disease mechanisms are still incompletely understood and this hampers finding an effective treatment. As NPC patients, who carry NPC1 mutations, have shown to share several pathological features with Alzheimer's disease (AD) and we and others have previously shown that AD is associated with a dysfunctionality of the blood-cerebrospinal fluid (CSF) barrier located at choroid plexus, we investigated the functionality of this latter barrier in NPC1 pathology. Using NPC1-/- mice, we show that despite an increase in inflammatory gene expression in choroid plexus epithelial (CPE) cells, the blood-CSF barrier integrity is not dramatically affected. Interestingly, we did observe a massive increase in autophagosomes in CPE cells and enlarged extracellular vesicles (EVs) in CSF upon NPC1 pathology. Additionally, we revealed that these EVs exert toxic effects on brain tissue, in vitro as well as in vivo. Moreover, we observed that EVs derived from the supernatant of NPC1-/- choroid plexus explants are able to induce typical brain pathology characteristics of NPC1-/-, more specifically microgliosis and astrogliosis. Taken together, our data reveal for the first time that the choroid plexus and CSF EVs might play a role in the brain-related pathogenesis of NPC1.
    Keywords:  Niemann-Pick type C disease; autophagosomes; blood-CSF-barrier; choroid plexus; extracellular vesicles
    DOI:  https://doi.org/10.3389/fncel.2021.757482
  25. Adv Biol Regul. 2021 Oct 08. pii: S2212-4926(21)00048-8. [Epub ahead of print]82 100832
    Phagosome maturation in macrophages: Eat, digest, adapt, and repeat.
    Aaron Fountain, Subothan Inpanathan, Patris Alves, Munira B Verdawala, Roberto J Botelho.
      Phagocytosis is a dynamic process that requires an intricate interplay between phagocytic receptors, membrane lipids, and numerous signalling proteins and their effectors, to coordinate the engulfment of a bound particle. These particles are diverse in their physico-chemical properties such as size and shape and include bacteria, fungi, apoptotic cells, living tumour cells, and abiotic particles. Once engulfed, these particles are enclosed within a phagosome, which undergoes a striking transformation referred to as phagosome maturation, which will ultimately lead to the processing and degradation of the enclosed particulate. In this review, we focus on recent advancements in phagosome maturation in macrophages, highlighting new discoveries and emerging themes. Such advancements include identification of new GTPases and their effectors and the intricate spatio-temporal dynamics of phosphoinositides in governing phagosome maturation. We then explore phagosome fission and recycling, the emerging role of membrane contact sites, and delve into mechanisms of phagosome resolution to recycle and reform lysosomes. We further illustrate how phagosome maturation is context-dependent, subject to the type of particle, phagocytic receptors, the phagocytes and their state of activation during phagocytosis. Lastly, we discuss how phagosomes serve as signalling platforms to help phagocytes adapt to their environmental conditions. Overall, this review aims to cover recent findings, identify emerging themes, and highlight current challenges and directions to improve our understanding of phagosome maturation in macrophages.
    Keywords:  Adaptation; Immunity; Membrane; Organelles; Phagocytosis; Signalling
    DOI:  https://doi.org/10.1016/j.jbior.2021.100832
  26. Arterioscler Thromb Vasc Biol. 2021 Nov 04. ATVBAHA121316552
    Early Endosomal GTPase Rab5 Regulates GPIb Trafficking and Platelet Production In Vitro.
    Ivana Bertović, Roberta Kurelić, Hana Mahmutefendić Lučin, Antonija Jurak Begonja.
       OBJECTIVE: Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3-monophosphate production important for binding of effector proteins during early-to-late endosomal/lysosomal maturation. Approach and Results: To investigate the role of Rab5 in megakaryocytes, we expressed GFP (green fluorescent protein)-coupled Rab5 wild type and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived megakaryocytes. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing megakaryocytes, when compared with GFP or Rab5 wild type. Moreover, trafficking of GPIbß, a subunit of major megakaryocytes receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbß was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 wild type megakaryocytes, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbß plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in late endosomal/lysosomal. Localization of GPIbß in enlarged endosomes was phosphatidylinositol 3-monophosphate dependent.
    CONCLUSIONS: Taken together, our results demonstrate that Rab5-dependent endocytosis plays an important role in megakaryocytes receptor trafficking, membrane formation, and thrombopoiesis.
    Keywords:  blood platelets; bone marrow; endosomes; thrombopoiesis; transferrin
    DOI:  https://doi.org/10.1161/ATVBAHA.121.316552
  27. Nat Commun. 2021 Nov 01. 12(1): 6267
    Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.
    Raed Ibraheim, Phillip W L Tai, Aamir Mir, Nida Javeed, Jiaming Wang, Tomás C Rodríguez, Suk Namkung, Samantha Nelson, Eraj Shafiq Khokhar, Esther Mintzer, Stacy Maitland, Zexiang Chen, Yueying Cao, Emmanouela Tsagkaraki, Scot A Wolfe, Dan Wang, Athma A Pai, Wen Xue, Guangping Gao, Erik J Sontheimer.
      Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.
    DOI:  https://doi.org/10.1038/s41467-021-26518-y
  28. Proc Natl Acad Sci U S A. 2021 Nov 09. pii: e2109327118. [Epub ahead of print]118(45):
    Cryo-EM structures of PI3Kα reveal conformational changes during inhibition and activation.
    Xiao Liu, Su Yang, Jonathan R Hart, Yingna Xu, Xinyu Zou, Huibing Zhang, Qingtong Zhou, Tian Xia, Yan Zhang, Dehua Yang, Ming-Wei Wang, Peter K Vogt.
      Phosphoinositide 3-kinases (PI3Ks) are lipid kinases essential for growth and metabolism. Their aberrant activation is associated with many types of cancers. Here we used single-particle cryoelectron microscopy (cryo-EM) to determine three distinct conformations of full-length PI3Kα (p110α-p85α): the unliganded heterodimer PI3Kα, PI3Kα bound to the p110α-specific inhibitor BYL-719, and PI3Kα exposed to an activating phosphopeptide. The cryo-EM structures of unbound and of BYL-719-bound PI3Kα are in general accord with published crystal structures. Local deviations are presented and discussed. BYL-719 stabilizes the structure of PI3Kα, but three regions of low-resolution extra density remain and are provisionally assigned to the cSH2, BH, and SH3 domains of p85. One of the extra density regions is in contact with the kinase domain blocking access to the catalytic site. This conformational change indicates that the effects of BYL-719 on PI3Kα activity extend beyond competition with adenosine triphosphate (ATP). In unliganded PI3Kα, the DFG motif occurs in the "in" and "out" positions. In BYL-719-bound PI3Kα, only the DFG-in position, corresponding to the active conformation of the kinase, was observed. The phosphopeptide-bound structure of PI3Kα is composed of a stable core resolved at 3.8 Å. It contains all p110α domains except the adaptor-binding domain (ABD). The p85α domains, linked to the core through the ABD, are no longer resolved, implying that the phosphopeptide activates PI3Kα by fully releasing the niSH2 domain from binding to p110α. The structures presented here show the basal form of the full-length PI3Kα dimer and document conformational changes related to the activated and inhibited states.
    Keywords:  activation; activity-dependent conformational changes; inhibition; phosphoinositide 3-kinase (PI3K)
    DOI:  https://doi.org/10.1073/pnas.2109327118
  29. Nat Commun. 2021 Nov 03. 12(1): 6354
    The EMT activator ZEB1 accelerates endosomal trafficking to establish a polarity axis in lung adenocarcinoma cells.
    Priyam Banerjee, Guan-Yu Xiao, Xiaochao Tan, Veronica J Zheng, Lei Shi, Maria Neus Bota Rabassedas, Hou-Fu Guo, Xin Liu, Jiang Yu, Lixia Diao, Jing Wang, William K Russell, Jason Roszik, Chad J Creighton, Jonathan M Kurie.
      Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change.
    DOI:  https://doi.org/10.1038/s41467-021-26677-y
  30. FEBS Lett. 2021 Nov 06.
    The involvement of sodium in the function of the human amino acid transporter ASCT2.
    Tiziano Mazza, Mariafrancesca Scalise, Gilda Pappacoda, Lorena Pochini, Cesare Indiveri.
      ASCT2 is a membrane amino acid transporter with relevance to human physiology and pathology, such as cancer. Notwithstanding, the study on the ASCT2 transport cycle still has unknown aspects, such as the role of Na+ in this process. We investigate this issue using recombinant hASCT2 reconstituted in proteoliposomes. Changes in the composition of purification buffers show the crucial role of Na+ in ASCT2 functionality. The transport activity is abolished when Na+ is absent or substituted by Li+ or K+ in purification buffers. By employing a Na+ fluorometric probe, we measured an inwardly directed flux of Na+ and, by combining fluorometric and radiometric assays, determined a 2Na+ :1Gln stoichiometry. Kinetics of Na+ transport suggest that pH-sensitive residues are involved in Na+ binding/transport. Our results clarify the role of Na+ on human ASCT2 transporter activity.
    Keywords:  ASCT2; Proteoliposomes; SLC; fluorometric assay; kinetics; membrane transport
    DOI:  https://doi.org/10.1002/1873-3468.14224
  31. Cell Rep. 2021 Nov 02. pii: S2211-1247(21)01418-2. [Epub ahead of print]37(5): 109945
    The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7.
    Evgeniya Trofimenko, Yuta Homma, Mitsunori Fukuda, Christian Widmann.
      Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Besides possible recycling routes to the plasma membrane and various organelles, previously described endocytic pathways (e.g., clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway) all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs), we identify an endocytic pathway that moves material to nonacidic Lamp1-positive late endosomes. Trafficking via this endocytic route is fully independent of Rab5 and Rab7 but requires the Rab14 protein. The pathway taken by CPPs differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already, as it is not affected by a series of compounds that inhibit macropinocytosis or clathrin-mediated endocytosis. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins.
    Keywords:  Lamp1; Rab14; Rab5; Rab7; cell-penetrating peptides; endocytosis; endosomes; homeodomains; homeoproteins; polyamines
    DOI:  https://doi.org/10.1016/j.celrep.2021.109945
  32. Trends Cardiovasc Med. 2021 Nov 01. pii: S1050-1738(21)00127-4. [Epub ahead of print]
    Clinical features of Danon disease and insights gained from LAMP-2 deficiency models.
    Yafei Zhai, Jinxin Miao, Ying Peng, Yaohe Wang, Jianzeng Dong, Xiaoyan Zhao.
      Danon disease (DD) is an X-linked multisystem disorder with clinical features characterized by the triad of hypertrophic cardiomyopathy, skeletal muscle weakness, and mental retardation. Cardiac involvement can be fatal in the absence of an effective treatment option such as heart transplantation. Molecular studies have proved that LAMP-2 protein deficiency, mainly LAMP-2B isoform, resulting from LAMP2 gene mutation, is the culprit for DD. Autophagy impairment due to LAMP-2 deficiency mediated the accumulation of abnormal autophagic vacuoles in cells. While it is not ideal for mimicking DD phenotypes in humans, the emergence of LAMP-2-deficient animal models and induced pluripotent stem cells from DD patients provided powerful tools for exploring DD mechanism. In both in vitro and in vivo studies, much evidence has demonstrated that mitochondria dysfunction and fragmentation can result in DD pathology. Fundamental research contributes to the therapeutic transformation. By targeting the molecular core, several potential therapies have demonstrated promising results in partial phenotypes improvement. Among them, gene therapies anticipate inaugurate a class of symptom control and prevention drugs as their in vivo effects are promising, and one clinical trial is currently underway.
    Keywords:  Animal models; Danon disease; LAMP2; iPSC
    DOI:  https://doi.org/10.1016/j.tcm.2021.10.012
  33. Nat Cell Biol. 2021 Nov 04.
    FGFR-inhibitor-mediated dismissal of SWI/SNF complexes from YAP-dependent enhancers induces adaptive therapeutic resistance.
    Yihao Li, Xintao Qiu, Xiaoqing Wang, Hui Liu, Renee C Geck, Alok K Tewari, Tengfei Xiao, Alba Font-Tello, Klothilda Lim, Kristen L Jones, Murry Morrow, Raga Vadhi, Pei-Lun Kao, Aliya Jaber, Smitha Yerrum, Yingtian Xie, Kin-Hoe Chow, Paloma Cejas, Quang-Dé Nguyen, Henry W Long, X Shirley Liu, Alex Toker, Myles Brown.
      How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodelling, leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters, resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Consistent with this mechanism, addition of mTORC1 or YAP inhibitors to FGFR blockade synergistically attenuated the growth of TNBC patient-derived xenograft models. Collectively, these findings reveal a feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance and provides potential therapeutic strategies to overcome this mechanism of resistance.
    DOI:  https://doi.org/10.1038/s41556-021-00781-z
  34. Cell Rep. 2021 Nov 02. pii: S2211-1247(21)01364-4. [Epub ahead of print]37(5): 109894
    SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase.
    Won Young Choi, Seongok Kim, Philipp Aurass, Wenwen Huo, Elizabeth A Creasey, Marc Edwards, Martin Lowe, Ralph R Isberg.
      Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.
    Keywords:  Legionella pneumophila; OCRL; bacterial pathogenesis; endosomes; intracellular replication; vesicle trafficking
    DOI:  https://doi.org/10.1016/j.celrep.2021.109894
  35. Sci Adv. 2021 Nov 05. 7(45): eabh1541
    Lipid-induced DRAM recruits STOM to lysosomes and induces LMP to promote exosome release from hepatocytes in NAFLD.
    Jie Zhang, Jie Tan, Mengke Wang, Yifen Wang, Mengzhen Dong, Xuefeng Ma, Baokai Sun, Shousheng Liu, Zhenzhen Zhao, Lizhen Chen, Kai Liu, Yongning Xin, Likun Zhuang.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/sciadv.abh1541
  36. Cancer Lett. 2021 Oct 30. pii: S0304-3835(21)00549-8. [Epub ahead of print]525 33-45
    Long noncoding RNA KB-1460A1.5 inhibits glioma tumorigenesis via miR-130a-3p/TSC1/mTOR/YY1 feedback loop.
    Lixia Xu, Qiaoli Wu, Hua Yan, Chang Shu, Weijia Fan, Xiaoguang Tong, Qingguo Li.
      Long noncoding RNAs (lncRNAs) have been shown to be closely related to cancer progression and therapy. However, the clinical significance of lncRNAs and the mechanisms by which they function in glioma are largely unknown. In this study, using online data sets combined with collected clinical glioma tissues, we determined that the lncRNA KB-1460A1.5 is downregulated and positively correlated with prognosis in glioma. Functional experiments showed that overexpression of KB-1460A1.5 inhibits glioma cell proliferation, migration and invasion in vitro and in vivo, while downregulation of KB-1460A1.5 has the opposite effects. Mechanistically, tandem mass tag (TMT)-based quantitative proteomic analysis revealed that KB-1460A1.5 preferentially affects the Akt/TSC1/mTOR pathway. KB-1460A1.5 was found to function as a competing endogenous RNA (ceRNA) to regulate the expression of TSC1, a key regulatory component of the mTOR pathway, by sponging miR-130a-3p in glioma cells. Furthermore, our data demonstrate that the mTOR pathway regulates the expression of the transcription factor Yin Yang 1 (YY1), which in turn binds directly to the KB-1460A1.5 promoter and affects the expression of KB-1460A1.5. Untargeted metabolomics and quantitative real-time PCR (qRT-PCR) analysis further confirmed the effects of KB-1460A1.5 on amino acid metabolism. In conclusion, this study revealed that lncRNA KB-1460A1.5 inhibits glioma tumorigenesis via miR-130a-3p/TSC1/mTOR/YY1 feedback loop.
    Keywords:  Amino acid metabolism; Glioma; KB-1460A1.5; TSC1; miR-130a-3p
    DOI:  https://doi.org/10.1016/j.canlet.2021.10.033
  37. Orphanet J Rare Dis. 2021 Oct 30. 16(1): 456
    Evaluation of the long-term treatment effects of intravenous idursulfase in patients with mucopolysaccharidosis II (MPS II) using statistical modeling: data from the Hunter Outcome Survey (HOS).
    Joseph Muenzer, Jaco Botha, Paul Harmatz, Roberto Giugliani, Christoph Kampmann, Barbara K Burton.
       BACKGROUND: Mucopolysaccharidosis II (MPS II; Hunter syndrome) is a rare, life-limiting lysosomal storage disease caused by deficient iduronate-2-sulfatase activity. Enzyme replacement therapy (ERT) with intravenous (IV) idursulfase can stabilize or improve many somatic manifestations, but there remains a need for further analysis of long-term treatment outcomes. Using data from patients with MPS II enrolled in the Hunter Outcome Survey (HOS), mixed modeling was performed to evaluate and predict the effects of IV idursulfase treatment on selected clinical parameters for up to 8 years following treatment start. The modeling population comprised male patients followed prospectively in HOS who had received IV idursulfase for at least 5 years and who had data available for two or more time points (at least one post-ERT). Age at ERT start and time since ERT start were included as covariates.
    RESULTS: In total, 481 patients were eligible for inclusion in at least one model. At 8 years post-ERT start, improvement from baseline was predicted for each age group (< 18 months, 18 months to < 5 years and ≥ 5 years at treatment start) in the following parameters: mean urinary glycosaminoglycan levels (percentage changes of > -75% in each group), mean left ventricular mass index (decreases of ~ 1 g/m2) and mean palpable liver size (decreases of > 2 cm). Improvements in mean 6-min walk test distance (increase of > 50 m) and stabilization in percent predicted forced vital capacity and forced expiratory volume in 1 s (decreases of ~ 4 and ~ 9 percentage points, respectively) at 8 years post-ERT start were predicted for patients aged ≥ 5 years at ERT start (these assessments are unsuitable for patients aged < 5 years). Predicted changes over time were similar across the three age groups; however, overall outcomes were most favorable in children aged < 18 months at ERT start.
    CONCLUSIONS: These findings suggest that the previously reported positive effects of IV idursulfase on the somatic manifestations of MPS II are predicted to be maintained for at least 8 years following ERT initiation and highlight the value of statistical modeling to predict long-term treatment outcomes in patients with rare diseases.
    Keywords:  Disease registry; Enzyme replacement therapy; Hunter syndrome; Idursulfase; Lysosomal storage disease; MPS II; Mucopolysaccharidosis II; Statistical modeling
    DOI:  https://doi.org/10.1186/s13023-021-02052-4
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