bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒08‒23
fifty-one papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth.
  2. Apolipoprotein D-mediated preservation of lysosomal function promotes cell survival and delays motor impairment in Niemann-Pick type A disease.
  3. Amino acid-dependent control of mTORC1 signaling: a variety of regulatory modes.
  4. Complement modulation reverses pathology in Y402H-retinal pigment epithelium cell model of age-related macular degeneration by restoring lysosomal function.
  5. Rictor amplification promotes NSCLC cell proliferation through formation and activation of mTORC2 at the expense of mTORC1.
  6. TMEM175 mediates Lysosomal function and participates in neuronal injury induced by cerebral ischemia-reperfusion.
  7. A Comparative Effectiveness Study of Newborn Screening Methods for Four Lysosomal Storage Disorders.
  8. Inhibition of the mechanistic target of rapamycin induces cell survival via MAPK in tuberous sclerosis complex.
  9. Lyso-glycosphingolipids: presence and consequences.
  10. LRRK2 to the rescue of damaged endomembranes.
  11. Highlights on Genomics Applications for Lysosomal Storage Diseases.
  12. Altered level of plasma exosomes in patients with gaucher disease.
  13. mTOR-coordinated Post-Transcriptional Gene Regulations: from Fundamental to Pathogenic Insights.
  14. MiTF is Associated with Chemoresistance to Cisplatin in A549 Lung Cancer Cells via Modulating Lysosomal Biogenesis and Autophagy.
  15. Activated protein phosphatase 2A disrupts nutrient sensing balance between mTORC1 and AMPK causing sarcopenia in alcoholic liver disease.
  16. Liver steatosis is highly prevalent and is associated with metabolic risk factors and liver fibrosis in adult patients with type 1 Gaucher disease.
  17. Cathepsin K is a potent disaggregase of α-synuclein fibrils.
  18. A rare manifestation of right ventricular dysfunction in an adult patient with mucolipidosis type III α/β.
  19. LIN28B/let-7 control the ability of neonatal murine auditory supporting cells to generate hair cells through mTOR signaling.
  20. Novel Links between TORC1 and Traditional Non-Coding RNA, tRNA.
  21. An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission.
  22. An intramembrane chaperone complex facilitates membrane protein biogenesis.
  23. Loss-of-function variants in HOPS complex genes VPS16 and VPS41 cause early-onset dystonia associated with lysosomal abnormalities.
  24. TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress.
  25. Insights into DEPTOR regulation from in silico analysis of DEPTOR complexes.
  26. mTOR Inhibition Leads to Src-Mediated EGFR Internalisation and Degradation in Glioma Cells.
  27. Age-dependent loss of adipose Rubicon promotes metabolic disorders via excess autophagy.
  28. Lipofuscin in keratinocytes: production, properties, and consequences of the photosensitization with visible light.
  29. Unconventional role of lysosomes in phagocytosis.
  30. Targeting the ESCRT-III component CHMP2A for noncanonical Caspase-8 activation on autophagosomal membranes.
  31. The amino acid transporter Slc7a5 regulates the mTOR pathway and is required for granule cell development.
  32. The mechanism of activation of the actin binding protein EHBP1 by Rab8 family members.
  33. Nanotechnology-Based Targeting of mTOR Signaling in Cancer.
  34. A DNA-Based FLIM Reporter for Simultaneous Quantification of Lysosomal pH and Ca2+ during Autophagy Regulation.
  35. Glycogen synthase kinase-3β inhibits tubular regeneration in acute kidney injury by a FoxM1-dependent mechanism.
  36. Adenovirus Reveals New Pathway for Cholesterol Egress from the Endolysosomal System.
  37. LRRK2 kinase inhibitors reduce alpha-synuclein in human neuronal cell lines with the G2019S mutation.
  38. The MAPK and AMPK signalings: interplay and implication in targeted cancer therapy.
  39. LYTACs Target Extracellular and Membrane Proteins for Degradation.
  40. TonEBP Promotes β-Cell Survival under ER Stress by Enhancing Autophagy.
  41. Oncology Therapeutics Targeting the Metabolism of Amino Acids.
  42. Predicting the Development of Anti-Drug Antibodies against Recombinant alpha-Galactosidase A in Male Patients with Classical Fabry Disease.
  43. Amino acid homeostatic control by TORC1 in Saccharomyces cerevisiae under high hydrostatic pressure.
  44. Analysis of the alpha galactosidase gene: mutation profile and description of two novel mutations with extensive literature review in Turkish population.
  45. An overview of deregulated lipid metabolism in nonalcoholic fatty liver disease with special focus on lysosomal acid lipase.
  46. N-Terminal-Dependent Protein Degradation and Targeting Cancer Cells.
  47. Osteoclasts' Ability to Generate Trenches Rather Than Pits Depends on High Levels of Active Cathepsin K and Efficient Clearance of Resorption Products.
  48. Decoding three distinct states of the Syntaxin17 SNARE motif in mediating autophagosome-lysosome fusion.
  49. Age-induced accumulation of methylmalonic acid promotes tumour progression.
  50. Rictor promotes tumor progression of rapamycin-insensitive triple-negative breast cancer cells.
  51. BAP1 regulates AMPK-mTOR signalling pathway through deubiquitinating and stabilizing tumour-suppressor LKB1.
  1. Nat Cell Biol. 2020 Aug 17.
    p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth.
    Ada Nowosad, Pauline Jeannot, Caroline Callot, Justine Creff, Renaud Thierry Perchey, Carine Joffre, Patrice Codogno, Stephane Manenti, Arnaud Besson.
      Autophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies reported that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy by an unknown mechanism. Here we find that p27 controls autophagy via an mTORC1-dependent mechanism in amino acid-deprived cells. During prolonged starvation, a fraction of p27 is recruited to lysosomes, where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 activation. Binding of p27 to LAMTOR1 prevents Ragulator assembly and mTORC1 activation, promoting autophagy. Conversely, p27-/- cells exhibit elevated mTORC1 signalling as well as impaired lysosomal activity and autophagy. This is associated with cytoplasmic sequestration of TFEB, preventing induction of the lysosomal genes required for lysosome function. LAMTOR1 silencing or mTOR inhibition restores autophagy and induces apoptosis in p27-/- cells. Together, these results reveal a direct coordinated regulation between the cell cycle and cell growth machineries.
    DOI:  https://doi.org/10.1038/s41556-020-0554-4
  2. Neurobiol Dis. 2020 Aug 12. pii: S0969-9961(20)30321-1. [Epub ahead of print] 105046
    Apolipoprotein D-mediated preservation of lysosomal function promotes cell survival and delays motor impairment in Niemann-Pick type A disease.
    Raquel Pascua-Maestro, Miriam Corraliza-Gomez, Cristian Fadrique-Rojo, Maria D Ledesma, Edward H Schuchman, Diego Sanchez, Maria D Ganfornina.
      Lysosomal Storage Diseases (LSD) are genetic diseases causing systemic and nervous system dysfunction. The glia-derived lipid binding protein Apolipoprotein D (ApoD) is required for lysosomal functional integrity in glial and neuronal cells, ensuring cell survival upon oxidative stress or injury. Here we test whether ApoD counteracts the pathogenic consequences of a LSD, Niemann Pick-type-A disease (NPA), where mutations in the acid sphingomyelinase gene result in sphingomyelin accumulation, lysosomal permeabilization and early-onset neurodegeneration. We performed a multivariable analysis of behavioral, cellular and molecular outputs in 12 and 24 week-old male and female NPA model mice, combined with ApoD loss-of-function mutation. Lack of ApoD in NPA mice accelerates cerebellar-dependent motor deficits, enhancing loss of Purkinje neurons. We studied ApoD expression in brain sections from a NPA patient and age-matched control, and the functional consequences of ApoD supplementation in primary human fibroblasts from two independent NPA patients and two control subjects. Cell viability, lipid peroxidation, and lysosomal functional integrity (pH, Cathepsin B activity, Galectin-3 exclusion) were examined. ApoD is endogenously overexpressed in NPA patients and NPA mouse brains and targeted to lysosomes of NPA patient cells, including Purkinje neurons and cultured fibroblasts. The accelerated lysosomal targeting of ApoD by oxidative stress is hindered in NPA fibroblasts, contributing to NPA lysosomes vulnerability. Exogenously added ApoD reduces NPA-prompted lysosomal permeabilization and alkalinization, reverts lipid peroxides accumulation, and significantly increases NPA cell survival. ApoD administered simultaneously to sphingomyelin overload results in complete rescue of cell survival. Our results reveal that ApoD protection of lysosomal integrity counteracts NPA pathology. ApoD supplementation could significantly delay not only the progression of NPA disease, but also of other LSDs through its beneficial effects in lysosomal functional maintenance.
    Keywords:  Human NPA fibroblasts; Human brain; Lipid binding protein; Lipocalin; Lysosomal pH; Lysosomal storage disorder; Lysosome permeability; Motor behavior; Neuroprotection; Purkinje neurons
    DOI:  https://doi.org/10.1016/j.nbd.2020.105046
  3. J Biomed Sci. 2020 Aug 17. 27(1): 87
    Amino acid-dependent control of mTORC1 signaling: a variety of regulatory modes.
    Terunao Takahara, Yuna Amemiya, Risa Sugiyama, Masatoshi Maki, Hideki Shibata.
      The mechanistic target of rapamycin complex 1 (mTORC1) is an essential regulator of cell growth and metabolism through the modulation of protein and lipid synthesis, lysosome biogenesis, and autophagy. The activity of mTORC1 is dynamically regulated by several environmental cues, including amino acid availability, growth factors, energy levels, and stresses, to coordinate cellular status with environmental conditions. Dysregulation of mTORC1 activity is closely associated with various diseases, including diabetes, cancer, and neurodegenerative disorders. The discovery of Rag GTPases has greatly expanded our understanding of the regulation of mTORC1 activity by amino acids, especially leucine and arginine. In addition to Rag GTPases, other factors that also contribute to the modulation of mTORC1 activity have been identified. In this review, we discuss the mechanisms of regulation of mTORC1 activity by particular amino acids.
    Keywords:  Amino acids; Intracellular Ca2+ concentration; Rag GTPases; Rheb GTPase; mTOR; mTORC1
    DOI:  https://doi.org/10.1186/s12929-020-00679-2
  4. Stem Cells Transl Med. 2020 Aug 20.
    Complement modulation reverses pathology in Y402H-retinal pigment epithelium cell model of age-related macular degeneration by restoring lysosomal function.
    Edvinas Cerniauskas, Marzena Kurzawa-Akanbi, Long Xie, Dean Hallam, Marina Moya-Molina, Kathryn White, David Steel, Mary Doherty, Phil Whitfield, Jumana Al-Aama, Lyle Armstrong, David Kavanagh, John D Lambris, Viktor I Korolchuk, Claire Harris, Majlinda Lako.
      Age-related macular degeneration (AMD) is a multifactorial disease, which is characterized by loss of central vision, affecting one in three people by the age of 75. The Y402H polymorphism in the complement factor H (CFH) gene significantly increases the risk of AMD. We show that Y402H-AMD-patient-specific retinal pigment epithelium (RPE) cells are characterized by a significant reduction in the number of melanosomes, an increased number of swollen lysosome-like-vesicles with fragile membranes, Cathepsin D leakage into drusen-like deposits and reduced lysosomal function. The turnover of C3 is increased significantly in high-risk RPE cells, resulting in higher internalization and deposition of the Terminal Complement Complex C5b-9 at the lysosomes. Inhibition of C3 processing via the compstatin analogue Cp40 reverses the disease phenotypes by relieving the lysosomes of their overburden and restoring their function. These findings suggest that modulation of the complement system represents a useful therapeutic approach for AMD patients associated with complement dysregulation.
    Keywords:  C3; C3b; C5b-9; Y402H polymorphism; autophagy; complement activation; complement factor H; human induced pluripotent stem cells; lysosome; retinal pigment epithelium
    DOI:  https://doi.org/10.1002/sctm.20-0211
  5. Mol Cancer Res. 2020 Aug 14. pii: molcanres.0262.2020. [Epub ahead of print]
    Rictor amplification promotes NSCLC cell proliferation through formation and activation of mTORC2 at the expense of mTORC1.
    Laura C Kim, Christopher H Rhee, Jin Chen.
      Non-small cell lung cancer (NSCLC) is characterized by genomic alterations, yet a targetable mutation has not been discovered in nearly half of all patients. Recent studies have identified amplification of RICTOR, an mTORC2-specific cofactor, as a novel actionable target in NSCLC. mTORC2 is one of two distinct mTOR complexes to sense environmental cues and regulate a variety of cellular processes including cell growth, proliferation, and metabolism, all of which promote tumorigenesis when aberrantly regulated. Interestingly, other components of mTORC2 are not co-amplified with RICTOR in human lung cancer, raising the question as to whether RICTOR amplification-induced changes are dependent on mTORC2 function. To model RICTOR amplification, we overexpressed Rictor using the Cas9 Synergistic Activator system. Overexpression of Rictor increased mTORC2 integrity and signaling, but at the expense of mTORC1, suggesting that overexpressed Rictor recruits common components away from mTORC1. Additionally, Rictor overexpression increases proliferation and growth of NSCLC 3D cultures and tumors in vivo. Conversely, knockout of RICTOR leads to decreased mTORC2 formation and activity, but increased mTORC1 function. Because Rictor has mTOR-dependent and independent functions, we also knocked out mLST8, a shared mTOR co-factor but is specifically required for mTORC2 function. Inducible loss of mLST8 in RICTOR-amplified NSCLC cells inhibited mTORC2 integrity and signaling, tumor cell proliferation, and tumor growth. Collectively, these data identify a mechanism for Rictor-driven tumor progression and provide further rationale for development of an mTORC2-specific inhibitor. Implications: RICTOR amplification drives NSCLC proliferation through formation of mTORC2, suggesting mTORC2-specific inhibition could be a beneficial therapeutic option.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0262
  6. Mol Brain. 2020 Aug 15. 13(1): 113
    TMEM175 mediates Lysosomal function and participates in neuronal injury induced by cerebral ischemia-reperfusion.
    Mengling Zhang, Haifeng Lu, Xueshun Xie, Haitao Shen, Xiang Li, Yunhai Zhang, Jiang Wu, Jianqiang Ni, Haiying Li, Gang Chen.
      As the main organelles for the clearance of damaged proteins and damaged organelles, the function of lysosomes is crucial for maintaining the intracellular homeostasis of long-lived neurons. A stable acidic environment is essential for lysosomes to perform their functions. TMEM175 has been identified as a new K+ channel that is responsible for regulating lysosomal membrane potential and pH stability in neurons. This study aimed to understand the role of TMEM175 in lysosomal function of neurons and neuronal injury following cerebral ischemia-reperfusion (I/R). A middle-cerebral-artery occlusion/reperfusion (MCAO/R) model was established in adult male Sprague-Dawley rats in vivo, and cultured neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic ischemia-reperfusion (I/R) injury in vitro. We found that the protein level of TMEM175 decreased after cerebral I/R injury and that TMEM175 overexpression ameliorated MCAO/R-induced brain-cell death and neurobehavioral deficits in vivo. Furthermore, these results were recapitulated in cultured neurons. Acridine orange (AO) staining, as well as LysoSensor Green DND-189, cathepsin-B (CTSB), and cathepsin-D (CTSD) activities, showed that TMEM175 deficiency inhibited the hydrolytic function of lysosomes by affecting lysosomal pH. In contrast, TMEM175 upregulation reversed OGD/R-induced lysosomal dysfunction and impaired mitochondrial accumulation in cultured neurons. TMEM175 deficiency induced by cerebral I/R injury leads to compromised lysosomal pH stability, thus inhibiting the hydrolytic function of lysosomes. Consequently, lysosomal-dependent degradation of damaged mitochondria is suppressed and thereby exacerbates brain damage. Exogenous up-regulation of TMEM175 protein level could reverse the neuronal lysosomal dysfunction after ischemia-reperfusion.
    Keywords:  Ischemia-reperfusion injury; Ischemic stroke; Lysosome; Neuron; TMEM175
    DOI:  https://doi.org/10.1186/s13041-020-00651-z
  7. Int J Neonatal Screen. 2020 Jun;pii: 44. [Epub ahead of print]6(2):
    A Comparative Effectiveness Study of Newborn Screening Methods for Four Lysosomal Storage Disorders.
    Karen A Sanders, Dimitar K Gavrilov, Devin Oglesbee, Kimiyo M Raymond, Silvia Tortorelli, John J Hopwood, Fred Lorey, Ramanath Majumdar, Charles A Kroll, Amber M McDonald, Jean M Lacey, Coleman T Turgeon, Justin N Tucker, Hao Tang, Robert Currier, Grazia Isaya, Piero Rinaldo, Dietrich Matern.
      Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.
    Keywords:  Fabry disease; Gaucher disease; Pompe disease; bioinformatics; immunoassay; microfluidics; mucopolysaccharidosis type I; newborn screening; post-analytical interpretation; tandem mass spectrometry
    DOI:  https://doi.org/10.3390/ijns6020044
  8. Orphanet J Rare Dis. 2020 Aug 17. 15(1): 209
    Inhibition of the mechanistic target of rapamycin induces cell survival via MAPK in tuberous sclerosis complex.
    Yiyang Lu, Erik Y Zhang, Jie Liu, Jane J Yu.
      BACKGROUND: Tuberous sclerosis complex (TSC) is a genetic disorder that cause tumors to form in many organs. These lesions may lead to epilepsy, autism, developmental delay, renal, and pulmonary failure. Loss of function mutations in TSC1 and TSC2 genes by aberrant activation of the mechanistic target of rapamycin (mTORC1) signaling pathway are the known causes of TSC. Therefore, targeting mTORC1 becomes a most available therapeutic strategy for TSC. Although mTORC1 inhibitor rapamycin and Rapalogs have demonstrated exciting results in the recent clinical trials, however, tumors rebound and upon the discontinuation of the mTORC1 inhibition. Thus, understanding the underlying molecular mechanisms responsible for rapamycin-induced cell survival becomes an urgent need. Identification of additional molecular targets and development more effective remission-inducing therapeutic strategies are necessary for TSC patients.RESULTS: We have discovered an Mitogen-activated protein kinase (MAPK)-evoked positive feedback loop that dampens the efficacy of mTORC1 inhibition. Mechanistically, mTORC1 inhibition increased MEK1-dependent activation of MAPK in TSC-deficient cells. Pharmacological inhibition of MAPK abrogated this feedback loop activation. Importantly, the combinatorial inhibition of mTORC1 and MAPK induces the death of TSC2-deficient cells.
    CONCLUSIONS: Our results provide a rationale for dual targeting of mTORC1 and MAPK pathways in TSC and other mTORC1 hyperactive neoplasm.
    Keywords:  Cell survival; Lymangioleiomyomatosis; MAPK signaling pathway; Rapamycin; Tuberous sclerosis complex; Tumor progression
    DOI:  https://doi.org/10.1186/s13023-020-01490-w
  9. Essays Biochem. 2020 Aug 18. pii: EBC20190090. [Epub ahead of print]
    Lyso-glycosphingolipids: presence and consequences.
    Marco van Eijk, Maria J Ferraz, Rolf G Boot, Johannes M F G Aerts.
      Lyso-glycosphingolipids are generated in excess in glycosphingolipid storage disorders. In the course of these pathologies glycosylated sphingolipid species accumulate within lysosomes due to flaws in the respective lipid degrading machinery. Deacylation of accumulating glycosphingolipids drives the formation of lyso-glycosphingolipids. In lysosomal storage diseases such as Gaucher Disease, Fabry Disease, Krabbe disease, GM1 -and GM2 gangliosidosis, Niemann Pick type C and Metachromatic leukodystrophy massive intra-lysosomal glycosphingolipid accumulation occurs. The lysosomal enzyme acid ceramidase generates the deacylated lyso-glycosphingolipid species. This review discusses how the various lyso-glycosphingolipids are synthesized, how they may contribute to abnormal immunity in glycosphingolipid storing lysosomal diseases and what therapeutic opportunities exist.
    Keywords:  acid ceramidase; glycosphingolipid; immune response; lysosomal storage disease
    DOI:  https://doi.org/10.1042/EBC20190090
  10. EMBO J. 2020 Aug 16. e106162
    LRRK2 to the rescue of damaged endomembranes.
    Maja Radulovic, Harald Stenmark.
      Mutations in several genes encoding for lysosomal proteins are involved in Parkinson's disease (PD). In this issue, Herbst et al (2020) show that PD-related leucine-rich repeat kinase 2 (LRRK2) is activated in response to pathogen or membranolytic drug-induced damage of phagolysosomes and lysosomes in macrophages, and regulates endolysosomal homeostasis by controlling the balance between membrane repair and degradation.
    DOI:  https://doi.org/10.15252/embj.2020106162
  11. Cells. 2020 Aug 14. pii: E1902. [Epub ahead of print]9(8):
    Highlights on Genomics Applications for Lysosomal Storage Diseases.
    Valentina La Cognata, Maria Guarnaccia, Agata Polizzi, Martino Ruggieri, Sebastiano Cavallaro.
      Lysosomal storage diseases (LSDs) are a heterogeneous group of rare multisystem genetic disorders occurring mostly in infancy and childhood, characterized by a gradual accumulation of non-degraded substrates inside the lysosome. Although the cellular pathogenesis of LSDs is complex and still not fully understood, the approval of disease-specific therapies and the rapid emergence of novel diagnostic methods led to the implementation of extensive national newborn screening (NBS) programs in several countries. In the near future, this will help the development of standardized workflows aimed to more timely diagnose these conditions. Hereby, we report an overview of LSD diagnostic process and treatment strategies, provide an update on the worldwide NBS programs, and discuss the opportunities and challenges arising from genomics applications in screening, diagnosis, and research.
    Keywords:  diagnosis; genomics; lysosomal storage diseases; newborn screening
    DOI:  https://doi.org/10.3390/cells9081902
  12. Eur J Med Genet. 2020 Aug 18. pii: S1769-7212(20)30306-2. [Epub ahead of print] 104038
    Altered level of plasma exosomes in patients with gaucher disease.
    Shtam Tatiana, Naryzhny Stanislav, Kulabukhova Darya, Garaeva Luiza, Senkevich Konstantin, Landa Sergey, Varfolomeeva Elena, Salogub Galina, Verlov Nikolai, Kopylov Arthur, Zorina Elena, Kamyshinsky Roman, Usenko Tatiana, Schwarzman Alexander, Zakharova Ekaterina, Emelyanov Anton, Pchelina Sofya.
      Mutations in the glucocerebrosidase gene (GBA) cause Gaucher disease (GD), the lysosomal storage disorder (LSD), and are the most common genetic risk factor of Parkinson's disease (PD). Lysosome functionality plays a critical role for secretion of extracellular vesicles (EVs) and their content. Here we compared EVs from the blood plasma of 8 GD patients and 8 controls in terms of amounts, size distribution, and composition of their protein cargo. EVs were isolated via sequential centrifugation and characterized by сryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). The presence of exosomal markers HSP70 and tetrasponins were analyzed by Western blot and flow cytometry. Protein profiling was performed by mass-spectrometry (shotgun analysis). Here, for the first time we reported an increased size and altered morphology in exosomes derived from blood plasma of GD patients. An increased size of plasma exosomes from GD patients compared to controls was demonstrated by cryo-EM and DLS (р<0.0001, p < 0.001, respectively) and confirmed by mode size detected by NTA (p < 0.02). Cryo-EM demonstrated an increased number of double and multilayer vesicles in plasma EVs from GD patients. We found that the EVs were enriched with the surface exosomal markers (CD9, СD63, CD81) and an exosome-associated protein HSP70 in case of the patients with the disease. Proteomic profiling of exosomal proteins did not reveal any proteins associated with PD pathogenesis. Thus, we showed that lysosomal dysfunction in GD patients lead to a striking alteration of plasma exosomes in size and morphology.
    Keywords:  Exosomes; GBA mutations; Gaucher disease; Lysosomes; Proteomics
    DOI:  https://doi.org/10.1016/j.ejmg.2020.104038
  13. J Lipid Atheroscler. 2020 Jan;9(1): 8-22
    mTOR-coordinated Post-Transcriptional Gene Regulations: from Fundamental to Pathogenic Insights.
    Hsin-Sung Yeh, Jeongsik Yong.
      Post-transcriptional regulations of mRNA transcripts such as alternative splicing and alternative polyadenylation can affect the expression of genes without changing the transcript levels. Recent studies have demonstrated that these post-transcriptional events can have significant physiological impacts on various biological systems and play important roles in the pathogenesis of a number of diseases, including cancers. Nevertheless, how cellular signaling pathways control these post-transcriptional processes in cells are not very well explored in the field yet. The mammalian target of rapamycin complex 1 (mTORC1) pathway plays a key role in sensing cellular nutrient and energy status and regulating the proliferation and growth of cells by controlling various anabolic and catabolic processes. Dysregulation of mTORC1 pathway can tip the metabolic balance of cells and is associated with a number of pathological conditions, including various types of cancers, diabetes, and cardiovascular diseases. Numerous reports have shown that mTORC1 controls its downstream pathways through translational and/or transcriptional regulation of the expression of key downstream effectors. And, recent studies have also shown that mTORC1 can control downstream pathways via post-transcriptional regulations. In this review, we will discuss the roles of post-transcriptional processes in gene expression regulations and how mTORC1-mediated post-transcriptional regulations contribute to cellular physiological changes. We highlight post-transcriptional regulation as an additional layer of gene expression control by mTORC1 to steer cellular biology. These emphasize the importance of studying post-transcriptional events in transcriptome datasets for gaining a fuller understanding of gene expression regulations in the biological systems of interest.
    Keywords:  Alternative splicing; Gene expression; Mammalian target of rapamycin; Polyadenylation; Transcriptome
    DOI:  https://doi.org/10.12997/jla.2020.9.1.8
  14. Cancer Manag Res. 2020 ;12 6563-6573
    MiTF is Associated with Chemoresistance to Cisplatin in A549 Lung Cancer Cells via Modulating Lysosomal Biogenesis and Autophagy.
    Wei Li, Xia Qin, Bin Wang, Ge Xu, Jun Zhang, Xuejun Jiang, Chengzhi Chen, Feng Qiu, Zhen Zou.
      Background: Non-small cell lung carcinoma (NSCLC) is often fatal; advanced NSCLC has a 5-year survival rate less than 20%. Platinum-based chemotherapy, in particular, cis-diamminedichloroplatinum (II) (cisplatin or DDP), is employed for the treatment of NSCLC; however, the drug resistance occurs frequently. Autophagy is defined as the process of intracellular degradation of cytoplasmic materials in the lysosome; however, the correlation between autophagy and drug resistance remains controversial. Herein, we investigated the correlation between autophagy and cisplatin resistance and also explored the underlying mechanisms.Methods and Results: We demonstrated that DDP-resistant NSCLC A549 (A549/DDP) cells had higher autophagy activity in comparison with its parental A549 cells; DDP treatment induced a time- and dose-dependent decrease of autophagy. Intriguingly, inhibition of autophagy with pharmacological drugs or knockdown of ATG5 or Beclin-1 aggravated cell death induced by DDP treatment, indicating that autophagy played protective roles during DDP treatment. Further mechanistic investigation revealed that DDP treatment could decrease the mRNA expression level of key autophagy-related genes, such as ATG5, Beclin-1, and ATG7, suggesting DDP repressed autophagy at the transcriptional level. The MiTF/TFE family (including TFEB, TFE3, TFEC, and MiTF) were involved in nutrient sensing and organelle biogenesis, and specifically, the lysosomal biogenesis. We found that only MiTF was dramatically decreased upon DDP treatment, and also a profound decrease of lysosomal markers, LAMP-1 or LAMP-2, suggesting that MiTF was involved in the modulation of lysosomal biogenesis and, consequently, the autophagy. Moreover, the knockdown of MiTF resulted in more severe cell death in A549/DDP cells, indicting the substantial correlation between MiTF and cisplatin chemoresistance.
    Conclusion: Our study provides novel insights into the association between MiTF and DDP chemoresistance in NSCLC cells, and suggests targeting MiTF and/or autophagy might be a potential strategy for the reversal of DDP chemoresistance for NSCLC treatment.
    Keywords:  MiTF; NSCLC; autophagy; chemoresistance; cisplatin
    DOI:  https://doi.org/10.2147/CMAR.S255939
  15. Hepatology. 2020 Aug 16.
    Activated protein phosphatase 2A disrupts nutrient sensing balance between mTORC1 and AMPK causing sarcopenia in alcoholic liver disease.
    Gangarao Davuluri, Nicole Welch, Jinendiran Sekar, Mahesha Gangadhariah, Khaled Alsabbagh Alchirazi, Maradumane L Mohan, Avinash Kumar, Sashi Kant, Samjhana Thapaliya, McKenzie Stine, Megan R McMullen, Rebecca L McCullough, George R Stark, Laura E Nagy, Sathyamangla V Naga Prasad, Srinivasan Dasarathy.
      BACKGROUND AND AIMS: Despite the high clinical significance of sarcopenia in alcoholic cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and AMP kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown).APPROACH AND RESULTS: Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from alcoholic cirrhotics were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue ATP indicating a "pseudofed" state. We identified protein phosphatase 2A (PP2A) as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A (I2-PP2A) to PP2A resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild type mice were observed in PI3Kγ-/- mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations.
    CONCLUSIONS: Our study describes the mechanistic underpinnings for previously unrecognized ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.
    Keywords:  Signaling; ethanol; phosphoinositide-3-kinase-gamma; protein homeostasis; skeletal muscle
    DOI:  https://doi.org/10.1002/hep.31524
  16. Liver Int. 2020 Aug 18.
    Liver steatosis is highly prevalent and is associated with metabolic risk factors and liver fibrosis in adult patients with type 1 Gaucher disease.
    Fabio Nascimbeni, Simonetta Lugari, Elena Cassinerio, Irene Motta, Alessia Cavicchioli, Annalisa Dalla Salda, Serena Bursi, Salvatore Donatiello, Vincenzo Spina, Maria Domenica Cappellini, Pietro Andreone, Francesca Carubbi.
      BACKGROUND AND AIMS: Gaucher disease (GD) is associated with peculiar metabolic abnormalities (i.e. hypermetabolic state, peripheral insulin resistance, dyslipidemia), partially reverted by enzyme replacement therapy (ERT) at the expense of weight gain. Such metabolic alterations together with an unhealthy lifestyle acquired by an aging GD population may favour the development of liver steatosis. We aimed at evaluating the prevalence of significant liver steatosis and at identifying the factors associated with liver steatosis in a cohort of patients with type 1 GD.METHODS: 20 adult type 1 GD patients from an Italian academic referral centre were prospectively submitted to vibration controlled transient elastography (Fibroscan®) with controlled attenuation parameter (CAP); significant steatosis was defined as CAP values ≥ 250 dB/min.
    RESULTS: Median CAP values were 234 [165-358] dB/min and 8 patients (40%) had significant steatosis. Significant steatosis was associated with indices of adiposity (weight, BMI and waist circumference), high blood pressure, insulin resistance and metabolic syndrome. GD-related variables and dose and duration of ERT were not associated with significant steatosis. In the subgroup of 16 patients on stable ERT for at least 24 months, CAP resulted significantly and positively associated with liver stiffness (rho 0.559, p=0.024).
    CONCLUSIONS: Significant steatosis is highly prevalent in adult type 1 GD patients and is strongly associated with a worse metabolic profile, featuring metabolic dysfunction-associated fatty liver disease (MAFLD). MAFLD may determine liver fibrosis progression in GD patients on stable ERT and may be a risk factor for long-term liver-related complications. EWC: 244.
    Keywords:  Controlled attenuation parameter; Enzyme replacement therapy; Glucocerebrosidase deficiency; Liver stiffness; Metabolic dysfunction-associated fatty liver disease
    DOI:  https://doi.org/10.1111/liv.14640
  17. Biochem Biophys Res Commun. 2020 Sep 03. pii: S0006-291X(20)31373-5. [Epub ahead of print]529(4): 1106-1111
    Cathepsin K is a potent disaggregase of α-synuclein fibrils.
    Ryan P McGlinchey, Shannon M Lacy, Robert L Walker, Jennifer C Lee.
      The intracellular accumulation of α-synuclein (α-syn) amyloid fibrils is a hallmark of Parkinson's disease. Because lysosomes are responsible for degrading aggregated species, enhancing lysosomal function could alleviate the overburden of α-syn. Previously, we showed that cysteine cathepsins (Cts) is the main class of lysosomal proteases that degrade α-syn, and in particular, CtsL was found to be capable of digesting α-syn fibrils. Here, we report that CtsK is a more potent protease for degrading α-syn amyloids. Using peptide mapping by liquid chromatography with mass spectrometry, critical cleavage sites involved in destabilizing fibril structure are identified. CtsK is only able to devour the internal regions after the removal of both N- and C-termini, indicating their protective role of the amyloid core from proteolytic attack. Our results suggest that if overexpressed in lysosomes, CtsK has the potential to ameliorate α-syn pathology.
    Keywords:  Cysteine cathepsin; LC-MS; Parkinson’s disease; α-synuclein
    DOI:  https://doi.org/10.1016/j.bbrc.2020.06.155
  18. Can J Cardiol. 2020 Aug 17. pii: S0828-282X(20)30909-0. [Epub ahead of print]
    A rare manifestation of right ventricular dysfunction in an adult patient with mucolipidosis type III α/β.
    Soichiro Kashihara, Kisho Ohtani, Tasuku Sato, Akiko Nishizaki, Yoko Shojima, Yuko Deguchi, Takeo Fujino, Toru Hashimoto, Taiki Higo, Hiroyuki Tsutsui.
      Mucolipidosis type III (ML III) α/β is an autosomal recessive lysosomal storage disease, caused by the deficient activity of UDP-N-acetyl glucosamine-1-phosphotransferase (GlcNAc-PTase). The resultant intra-lysosomal accumulation of partly degraded mucopolysaccharides and sphingolipids causes multiple organ damages including the heart. The most documented cardiac manifestation is the thickening and insufficiency of mitral and aortic valves, but there are very few reports about the myocardial involvement. We report a case with ML III α/β complicated by marked dilatation and dysfunction of the right ventricle (RV), which is quite rare and further broadens the clinical spectrum of the disease.
    DOI:  https://doi.org/10.1016/j.cjca.2020.07.239
  19. Proc Natl Acad Sci U S A. 2020 Aug 21. pii: 202000417. [Epub ahead of print]
    LIN28B/let-7 control the ability of neonatal murine auditory supporting cells to generate hair cells through mTOR signaling.
    Xiao-Jun Li, Angelika Doetzlhofer.
      Mechano-sensory hair cells within the inner ear cochlea are essential for the detection of sound. In mammals, cochlear hair cells are only produced during development and their loss, due to disease or trauma, is a leading cause of deafness. In the immature cochlea, prior to the onset of hearing, hair cell loss stimulates neighboring supporting cells to act as hair cell progenitors and produce new hair cells. However, for reasons unknown, such regenerative capacity (plasticity) is lost once supporting cells undergo maturation. Here, we demonstrate that the RNA binding protein LIN28B plays an important role in the production of hair cells by supporting cells and provide evidence that the developmental drop in supporting cell plasticity in the mammalian cochlea is, at least in part, a product of declining LIN28B-mammalian target of rapamycin (mTOR) activity. Employing murine cochlear organoid and explant cultures to model mitotic and nonmitotic mechanisms of hair cell generation, we show that loss of LIN28B function, due to its conditional deletion, or due to overexpression of the antagonistic miRNA let-7g, suppressed Akt-mTOR complex 1 (mTORC1) activity and renders young, immature supporting cells incapable of generating hair cells. Conversely, we found that LIN28B overexpression increased Akt-mTORC1 activity and allowed supporting cells that were undergoing maturation to de-differentiate into progenitor-like cells and to produce hair cells via mitotic and nonmitotic mechanisms. Finally, using the mTORC1 inhibitor rapamycin, we demonstrate that LIN28B promotes supporting cell plasticity in an mTORC1-dependent manner.
    Keywords:  LIN28; hair cell regeneration; inner ear cochlea; let-7 miRNA; mTOR pathway
    DOI:  https://doi.org/10.1073/pnas.2000417117
  20. Genes (Basel). 2020 Aug 19. pii: E956. [Epub ahead of print]11(9):
    Novel Links between TORC1 and Traditional Non-Coding RNA, tRNA.
    Yoko Otsubo, Yoshiaki Kamada, Akira Yamashita.
      Target of rapamycin (TOR) is a serine/threonine kinase that modulates cell growth and metabolism in response to environmental changes. Transfer RNA (tRNA) is an abundant and ubiquitous small non-coding RNA that is essential in the translation of mRNAs. Beyond its canonical role, it has been revealed that tRNAs have more diverse functions. TOR complex 1 (TORC1), which is one of the two TOR complexes, regulates tRNA synthesis by controlling RNA polymerase III. In addition to tRNA synthesis regulation, recent studies have revealed hidden connections between TORC1 and tRNA, which are both essential players in eukaryotic cellular activities. Here, we review the accumulating findings on the regulatory links between TORC1 and tRNA-particularly those links in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe.
    Keywords:  TORC1; budding yeast; fission yeast; tRNA
    DOI:  https://doi.org/10.3390/genes11090956
  21. Cell. 2020 Aug 13. pii: S0092-8674(20)30929-6. [Epub ahead of print]
    An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission.
    Anna-Katharina Pfitzner, Vincent Mercier, Xiuyun Jiang, Joachim Moser von Filseck, Buzz Baum, Anđela Šarić, Aurélien Roux.
      The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity.
    Keywords:  CHMP1; CHMP4; Did2; ESCRT; ESCRT-III; Ist1; Snf7; Vps2; in vitro reconstitution; membrane fission; membrane remodeling
    DOI:  https://doi.org/10.1016/j.cell.2020.07.021
  22. Nature. 2020 Aug 19.
    An intramembrane chaperone complex facilitates membrane protein biogenesis.
    Patrick J Chitwood, Ramanujan S Hegde.
      Integral membrane proteins are encoded by approximately 25% of all protein-coding genes1. In eukaryotes, the majority of membrane proteins are inserted, modified and folded at the endoplasmic reticulum (ER)2. Research over the past several decades has determined how membrane proteins are targeted to the ER and how individual transmembrane domains (TMDs) are inserted into the lipid bilayer3. By contrast, very little is known about how multi-spanning membrane proteins with several TMDs are assembled within the membrane. During the assembly of TMDs, interactions between polar or charged amino acids typically stabilize the final folded configuration4-8. TMDs with hydrophilic amino acids are likely to be chaperoned during the co-translational biogenesis of membrane proteins; however, ER-resident intramembrane chaperones are poorly defined. Here we identify the PAT complex, an abundant obligate heterodimer of the widely conserved ER-resident membrane proteins CCDC47 and Asterix. The PAT complex engages nascent TMDs that contain unshielded hydrophilic side chains within the lipid bilayer, and it disengages concomitant with substrate folding. Cells that lack either subunit of the PAT complex show reduced biogenesis of numerous multi-spanning membrane proteins. Thus, the PAT complex is an intramembrane chaperone that protects TMDs during assembly to minimize misfolding of multi-spanning membrane proteins and maintain cellular protein homeostasis.
    DOI:  https://doi.org/10.1038/s41586-020-2624-y
  23. Ann Neurol. 2020 Aug 18.
    Loss-of-function variants in HOPS complex genes VPS16 and VPS41 cause early-onset dystonia associated with lysosomal abnormalities.
    Dora Steel, Michael Zech, Chen Zhao, Katy Es Barwick, Derek Burke, Diane Demailly, Kishore R Kumar, Giovanna Zorzi, Nardo Nardocci, Rauan Kaiyrzhanov, Matias Wagner, Arcangela Iuso, Riccardo Berutti, Matej Škorvánek, Ján Necpál, Ryan Davis, Sarah Wiethoff, Kshitij Mankad, Sniya Sudhakar, Arianna Ferrini, Suvasini Sharma, Erik-Jan Kamsteeg, Marina A Tijssen, Corien Verschuuren, Martje E van Egmond, Joanna M Flowers, Meriel McEntagart, Arianna Tucci, , Philippe Coubes, Bernabe I Bustos, Paulina Gonzalez-Latapi, Stephen Tisch, Paul Darveniza, Kathleen M Gorman, Kathryn J Peall, Kai Bötzel, Jan C Koch, Tomasz Kmieć, Barbara Plecko, Sylvia Boesch, Bernhard Haslinger, Robert Jech, Barbara Garavaglia, Nick Wood, Henry Houlden, Paul Gissen, Steven J Lubbe, Carolyn M Sue, Laura Cif, Niccolò E Mencacci, Glenn Anderson, Manju A Kurian, Juliane Winkelmann.
      OBJECTIVES: The majority of people with suspected genetic dystonia remain undiagnosed after maximal investigation, implying that a number of causative genes have not yet been recognised. We aimed to investigate this paucity of diagnoses.METHODS: We undertook weighted burden analysis of whole-exome sequencing data from 138 individuals with unresolved generalised dystonia of suspected genetic aetiology, followed by additional case-finding from international databases, first for the gene implicated by the burden analysis (VPS16), then for other functionally related genes. Electron microscopy was performed on patient-derived cells.
    RESULTS: Analysis revealed a significant burden for VPS16 (Fisher's exact test p-value, 6.9x10-9 ). VPS16 encodes a subunit of the homotypic fusion and vacuole protein sorting (HOPS) complex, which plays a key role in autophagosome-lysosome fusion. A total of 18 individuals harbouring heterozygous loss-of-function VPS16 variants, and one with a microdeletion, were identified. These individuals experienced early-onset progressive dystonia with predominant cervical, bulbar, orofacial and upper limb involvement. Some patients had a more complex phenotype with additional neuropsychiatric and/or developmental comorbidities. We also identified biallelic loss-of-function variants in VPS41, another HOPS-complex encoding genes, in an individual with infantile-onset generalised dystonia. Electron microscopy of patient-derived lymphocytes and fibroblasts from both VPS16 and VPS41 patients showed vacuolar abnormalities suggestive of impaired lysosomal function.
    INTERPRETATION: Our study strongly supports a role for HOPS complex dysfunction in the pathogenesis of dystonia, though variants in different subunits display different phenotypic and inheritance characteristics. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1002/ana.25879
  24. Theranostics. 2020 ;10(20): 9280-9302
    TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress.
    Kailiang Zhou, Zhilong Zheng, Yao Li, Wen Han, Jing Zhang, Yuqin Mao, Huanwen Chen, Wanying Zhang, Mi Liu, Ling Xie, Hongyu Zhang, Huazi Xu, Jian Xiao.
      Background and Aim: Increasing evidence suggests that spinal cord injury (SCI)-induced defects in autophagic flux may contribute to an impaired ability for neurological repair following injury. Transcription factor E3 (TFE3) plays a crucial role in oxidative metabolism, lysosomal homeostasis, and autophagy induction. Here, we investigated the role of TFE3 in modulating autophagy following SCI and explored its impact on neurological recovery. Methods: Histological analysis via HE, Nissl and Mason staining, survival rate analysis, and behavioral testing via BMS and footprint analysis were used to determine functional recovery after SCI. Quantitative real-time polymerase chain reaction, Western blotting, immunofluorescence, TUNEL staining, enzyme-linked immunosorbent assays, and immunoprecipitation were applied to examine levels of autophagy flux, ER-stress-induced apoptosis, oxidative stress, and AMPK related signaling pathways. In vitro studies using PC12 cells were performed to discern the relationship between ROS accumulation and autophagy flux blockade. Results: Our results showed that in SCI, defects in autophagy flux contributes to ER stress, leading to neuronal death. Furthermore, SCI enhances the production of reactive oxygen species (ROS) that induce lysosomal dysfunction to impair autophagy flux. We also showed that TFE3 levels are inversely correlated with ROS levels, and increased TFE3 levels can lead to improved outcomes. Finally, we showed that activation of TFE3 after SCI is partly regulated by AMPK-mTOR and AMPK-SKP2-CARM1 signaling pathways. Conclusions: TFE3 is an important regulator in ROS-mediated autophagy dysfunction following SCI, and TFE3 may serve as a promising target for developing treatments for SCI.
    Keywords:  AMPK signaling pathways; Autophagy; ER stress-induced apoptosis; Spinal cord injury; TFE3
    DOI:  https://doi.org/10.7150/thno.46566
  25. J Struct Biol. 2020 Aug 13. pii: S1047-8477(20)30175-1. [Epub ahead of print] 107602
    Insights into DEPTOR regulation from in silico analysis of DEPTOR complexes.
    Aik-Hong Teh, Kean-Heng Yeap, Tamao Hisano.
      DEPTOR is an inhibitor of the mTOR kinase which controls cell growth. DEPTOR consists of two DEP domains and a PDZ domain connected by an unstructured linker, and its stability is tightly regulated through post-translational modifications of its linker region that contains the 286SSGYFS291 degron. Based on the mTORC1 complex, our modelling suggests a possible spatial arrangement of DEPTOR which is characterised to form a dimer. Our model shows that DEPTOR's two PDZ domains bind separately to the dimeric mTOR's FAT domains ∼13 Å apart, while the extended linkers are sufficiently long to span from the FAT domain to the kinase domain and beyond to join a shared dimer of the DEP domains. This places S299 closest to the kinase's catalytic site, indicating that phosphorylation would start with it and successively upstream towards the degron. The CK1α kinase is responsible for phosphorylation of DEPTOR's degron, and our docking analysis further reveals that CK1α contains sites to bind DEPTOR's pS286, pS287 and pT295, which may act as priming phosphates for the phosphorylation of S291 of DEPTOR's degron. DEPTOR's linker can also be ubiquitylated by the UbcH5A-SCFβ-TrCP complex without its PDZ dissociating from mTOR according to the modelling. As the catalytic cleft of mTOR's kinase is restricted, interactions between the kinase's unstructured segment surrounding the cleft and DEPTOR's linker, which may involve S293 and S299, may be critical in controlling DEPTOR's access to the catalytic cleft and hence its phosphorylation by mTOR in a manner dependent on mTOR's activation.
    Keywords:  DEPTOR; docking; homology modelling; kinase; mTOR
    DOI:  https://doi.org/10.1016/j.jsb.2020.107602
  26. Cancers (Basel). 2020 Aug 13. pii: E2266. [Epub ahead of print]12(8):
    mTOR Inhibition Leads to Src-Mediated EGFR Internalisation and Degradation in Glioma Cells.
    Barbara Colella, Mayra Colardo, Gianna Iannone, Claudia Contadini, Cristina Saiz-Ladera, Claudia Fuoco, Daniela Barilà, Guillermo Velasco, Marco Segatto, Sabrina Di Bartolomeo.
      Epidermal Growth Factor receptor (EGFR) is a tyrosine kinase receptor widely expressed on the surface of numerous cell types, which activates several downstream signalling pathways involved in cell proliferation, migration and survival. EGFR alterations, such as overexpression or mutations, have been frequently observed in several cancers, including glioblastoma (GBM), and are associated to uncontrolled cell proliferation. Here we show that the inhibition of mammalian target of Rapamycin (mTOR) mediates EGFR delivery to lysosomes for degradation in GBM cells, independently of autophagy activation. Coherently with EGFR internalisation and degradation, mTOR blockade negatively affects the mitogen activated protein/extracellular signal-regulated kinase (MAPK)/ERK pathway. Furthermore, we provide evidence that Src kinase activation is required for EGFR internaliation upon mTOR inhibition. Our results further support the hypothesis that mTOR targeting may represent an effective therapeutic strategy in GBM management, as its inhibition results in EGFR degradation and in proliferative signal alteration.
    Keywords:  EGFR; autophagy; glioma; mTOR
    DOI:  https://doi.org/10.3390/cancers12082266
  27. Nat Commun. 2020 Aug 18. 11(1): 4150
    Age-dependent loss of adipose Rubicon promotes metabolic disorders via excess autophagy.
    Tadashi Yamamuro, Tsuyoshi Kawabata, Atsunori Fukuhara, Shotaro Saita, Shuhei Nakamura, Hikari Takeshita, Mari Fujiwara, Yusuke Enokidani, Gota Yoshida, Keisuke Tabata, Maho Hamasaki, Akiko Kuma, Koichi Yamamoto, Iichiro Shimomura, Tamotsu Yoshimori.
      The systemic decline in autophagic activity with age impairs homeostasis in several tissues, leading to age-related diseases. A mechanistic understanding of adipocyte dysfunction with age could help to prevent age-related metabolic disorders, but the role of autophagy in aged adipocytes remains unclear. Here we show that, in contrast to other tissues, aged adipocytes upregulate autophagy due to a decline in the levels of Rubicon, a negative regulator of autophagy. Rubicon knockout in adipocytes causes fat atrophy and hepatic lipid accumulation due to reductions in the expression of adipogenic genes, which can be recovered by activation of PPARγ. SRC-1 and TIF2, coactivators of PPARγ, are degraded by autophagy in a manner that depends on their binding to GABARAP family proteins, and are significantly downregulated in Rubicon-ablated or aged adipocytes. Hence, we propose that age-dependent decline in adipose Rubicon exacerbates metabolic disorders by promoting excess autophagic degradation of SRC-1 and TIF2.
    DOI:  https://doi.org/10.1038/s41467-020-17985-w
  28. Free Radic Biol Med. 2020 Aug 15. pii: S0891-5849(20)31197-7. [Epub ahead of print]
    Lipofuscin in keratinocytes: production, properties, and consequences of the photosensitization with visible light.
    Paulo N Tonolli, Waleska K Martins, Helena C Junqueira, Maryana N Silva, Divinomar Severino, Carolina Santacruz-Perez, I Watanabe, Mauricio S Baptista.
      A dysfunction in the mitochondrial-lysosomal axis of cellular homeostasis is proposed to cause cells to age quicker and to accumulate lipofuscin. Typical protocols to mediate lipofuscinogenesis are based on the induction of the senescent phenotype either by allowing many consecutive cycles of cell division or by treating cells with physical/chemical agents such as ultraviolet (UV) light or hydrogen peroxide. Due to a direct connection with the physiopathology of age-related macular degeneration, lipofuscin that accumulates in retinal pigment epithelium (RPE) cells have been extensively studied, and the photochemical properties of RPE lipofuscin are considered as standard for this pigment. Yet, many other tissues such as the brain and the skin may prompt lipofuscinogenesis, and the properties of lipofuscin granules accumulated in these tissues are not necessarily the same as those of RPE lipofuscin. Here, we present a light-induced protocol that accelerates cell aging as judged by lipofuscinogenesis and maximizes this lipofuscinogenesis. Photosensitization of cells previously incubated with nanomolar concentrations of 1,9-dimethyl methylene blue (DMMB), severely and specifically damages mitochondria and lysosomes, leading to a lipofuscin-related senescent phenotype. By applying this protocol in human immortalized non-malignant keratinocytes (HaCaT) cells, we observed a 2.5-fold higher level of lipofuscin accumulation compared to the level of lipofuscin accumulation in cells treated with a typical UV protocol. Lipofuscin accumulated in keratinocytes exhibited the typical red light emission, with excitation maximum in the blue wavelength region (∼450 nm). Fluorescence lifetime image microscopy data showed that the keratinocyte lipofuscin has an emission lifetime of ∼ 1.7 ns. Lipofuscin-loaded cells (but not control cells) generated a substantial amount of singlet oxygen (1O2) when irradiated with blue light (420 nm), but there was no 1O2 generation when excitation was performed with a green light (532 nm). These characteristics were compared with those of RPE cells, considering that keratinocyte lipofuscin lacks the bisretinoids derivatives present in RPE lipofuscin. Additionally, we showed that lipofuscin-loaded keratinocytes irradiated with visible light presented critical DNA damages, such as double-strand breaks and Fpg-sensitive sites. We propose that the DMMB protocol is an efficient way to disturb the mitochondrial-lysosomal axis of cellular homeostasis, and consequently, it can be used to accelerate aging and to induce lipofuscinogenesis. We also discuss the consequences of the lipofuscin-induced genotoxicity of visible light in keratinocytes.
    Keywords:  DNA damage; autophagy; mitophagy; photoprotection; singlet oxygen; skin; visible light
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2020.08.002
  29. Cell Calcium. 2020 Aug 17. pii: S0143-4160(20)30111-1. [Epub ahead of print]91 102269
    Unconventional role of lysosomes in phagocytosis.
    Johannes Westman, Spencer A Freeman, Sergio Grinstein.
      Lysosomes are generally thought to be required only for the late stages of phagosome maturation, providing the proton pumps (V-ATPases) and hydrolases needed to acidify and degrade the ingested prey. A recent paper by Davis et al. (EMBO J. [2020], doi:10.15252/embj.2019104058) reports the involvement of lysosomes at a much earlier stage, namely in scission of phagosomes from the plasma membrane. Here we analyze these findings, highlighting a number of unexpected observations and unresolved questions.
    Keywords:  Calcineurin; Calcium; Dynamin; Lysosome; Macrophage; Phagocytosis; TPC; TRPML1
    DOI:  https://doi.org/10.1016/j.ceca.2020.102269
  30. Cell Death Differ. 2020 Aug 17.
    Targeting the ESCRT-III component CHMP2A for noncanonical Caspase-8 activation on autophagosomal membranes.
    Tatsuya Hattori, Yoshinori Takahashi, Longgui Chen, Zhenyuan Tang, Carson A Wills, Xinwen Liang, Hong-Gang Wang.
      Autophagosomal membranes can serve as activation platforms for intracellular death-inducing signaling complexes (iDISCs) to initiate Caspase-8-dependent apoptosis. In this study, we explore the impact of ESCRT-III-dependent phagophore closure on iDISC assemblies and cell death in osteosarcoma and neuroblastoma cells. Inhibition of phagophore closure by conditional depletion of CHMP2A, an ESCRT-III component, stabilizes iDISCs on immature autophagosomal membranes and induces Caspase-8-dependent cell death. Importantly, suppression of the iDISC formation via deletion of ATG7, an E1 enzyme for ubiquitin-like autophagy-related proteins, blocks Caspase-8 activation and cell death following CHMP2A depletion. Although DR5 expression and TRAIL-induced apoptosis are enhanced in CHMP2A-depleted cells, the canonical extrinsic pathway of apoptosis is not responsible for the initiation of cell death by CHMP2A depletion. Furthermore, the loss of CHMP2A impairs neuroblastoma tumor growth associated with decreased autophagy and increased apoptosis in vivo. Together, these findings indicate that inhibition of the ESCRT-III-dependent autophagosome sealing process triggers noncanonical Caspase-8 activation and apoptosis, which may open new avenues for therapeutic targeting of autophagy in cancer.
    DOI:  https://doi.org/10.1038/s41418-020-00610-0
  31. Hum Mol Genet. 2020 Aug 21. pii: ddaa186. [Epub ahead of print]
    The amino acid transporter Slc7a5 regulates the mTOR pathway and is required for granule cell development.
    Aidan M Sokolov, Jennie C Holmberg, David M Feliciano.
      Pathogenic mutations in the solute carrier family 7 member 5 (SLC7A5) gene which encodes an amino acid transporter cause microcephaly and seizures, yet the mechanisms responsible for these phenotypes are unclear. Models have demonstrated that Slc7a5 deletion is embryonic lethal and that these embryos lack a fully formed telencephalon. This phenotype is similar to that of mammalian Target of Rapamycin (mTOR) protein kinase deletion or mTOR inhibition. Notably, in many cells, Slc7a5 import of amino acids is required to maintain mTOR activity. Slc7a5 is present within neurogenic regions during embryogenesis, is found in cultured neurons, and can modulate neuronal electrophysiological properties. However, Slc7a5 is also highly expressed within endothelial cells of the blood brain barrier where removal in conditional mice leads to severe behavioral defects and non-cell autonomous changes in neurons. Therefore, the extent that neural Slc7a5 is required for development is unclear. Here, subventricular zone neural stem cells that generate olfactory bulb granule cell (GC) neurons were electroporated with SLC7A5 or Slc7a5 shRNA encoding plasmids. Although early phases of neural development were unaltered, Slc7a5 knockdown effected late phases of GC dendrite maturation and survival. Slc7a5 knockdown also decreased mTOR pathway activity. Rheb, an mTOR activator, rescued the effect of Slc7a5 knockdown on mTOR pathway activity and dendrite arbors. The data presented here demonstrate that Slc7a5 is required for GC mTOR pathway activity, maturation, and survival which may help explain why Slc7a5 mutations prevent normal brain development and function.
    DOI:  https://doi.org/10.1093/hmg/ddaa186
  32. Nat Commun. 2020 Aug 21. 11(1): 4187
    The mechanism of activation of the actin binding protein EHBP1 by Rab8 family members.
    Amrita Rai, Nathalie Bleimling, Ingrid R Vetter, Roger S Goody.
      EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.
    DOI:  https://doi.org/10.1038/s41467-020-17792-3
  33. Int J Nanomedicine. 2020 ;15 5767-5781
    Nanotechnology-Based Targeting of mTOR Signaling in Cancer.
    Mee-Sup Yoon.
      Mammalian target of rapamycin (mTOR) is a master regulator of cell growth and metabolism, which is activated in response to intra- and extracellular signals, including nutrients, growth factors, and cellular energy levels. The frequent dysregulation of mTOR signaling in cancer makes it an attractive therapeutic target, and several types of mTOR inhibitors have been developed. Nanoparticle-based mTOR modulators are predicted to target various cancers and deliver as well as release drugs in a controlled manner, resulting in enhanced bioavailability and reduced side effects. This mini-review is focused on the molecular mechanism of nanoparticle-based mTOR modulator action as well as the current development of mTOR inhibitors using nanoparticles. Understanding the biological function of nanoparticle-based mTOR modulators will contribute to the development of efficient nano-therapeutics for the treatment of cancers.
    Keywords:  RapaLinks; cancer; mTOR kinase inhibitor; mammalian target of rapamycin; nanoparticles; nanotechnology; rapalogs
    DOI:  https://doi.org/10.2147/IJN.S254574
  34. iScience. 2020 Aug 13. pii: S2589-0042(20)30628-3. [Epub ahead of print]23(9): 101436
    A DNA-Based FLIM Reporter for Simultaneous Quantification of Lysosomal pH and Ca2+ during Autophagy Regulation.
    Zhonghui Zhang, Zhichao Liu, Yang Tian.
      
    DOI:  https://doi.org/10.1016/j.isci.2020.101436
  35. FASEB J. 2020 Aug 19.
    Glycogen synthase kinase-3β inhibits tubular regeneration in acute kidney injury by a FoxM1-dependent mechanism.
    Sonali Sinha, Nidhi Dwivedi, James Woodgett, Shixin Tao, Christianna Howard, Timothy A Fields, Abeda Jamadar, Reena Rao.
      Acute kidney injury (AKI) is characterized by injury to the tubular epithelium that leads to the sudden loss of renal function. Proper tubular regeneration is essential to prevent progression to chronic kidney disease. In this study, we examined the role of FoxM1, a forkhead box family member transcription factor in tubular repair after AKI. Renal FoxM1 expression increased after renal ischemia/reperfusion (I/R)-induced AKI in mouse kidneys. Treatment with thiostrepton, a FoxM1 inhibitor, reduced FoxM1 regulated pro-proliferative factors and cell proliferation in vitro, and tubular regeneration in mouse kidneys after AKI. Glycogen synthase kinase-3 (GSK3) was found to be an upstream regulator of FoxM1 because GSK3 inhibition or renal tubular GSK3β gene deletion significantly increased FoxM1 expression, and improved tubular repair and renal function. GSK3 inactivation increased β-catenin, Cyclin D1, and c-Myc, and reduced cell cycle inhibitors p21 and p27. Importantly, thiostrepton treatment abolished the improved tubular repair in GSK3β knockout mice following AKI. These results demonstrate that FoxM1 is important for renal tubular regeneration following AKI and that GSK3β suppresses tubular repair by inhibiting FoxM1.
    Keywords:  FoxM1; GSK3β; acute kidney injury; cell proliferation; ischemia/reperfusion; thiostrepton; tubular regeneration
    DOI:  https://doi.org/10.1096/fj.202000526RR
  36. Int J Mol Sci. 2020 Aug 13. pii: E5808. [Epub ahead of print]21(16):
    Adenovirus Reveals New Pathway for Cholesterol Egress from the Endolysosomal System.
    Cathleen Carlin, Danny Manor.
      In addition to providing invaluable insights to the host response to viral infection, adenovirus continues to be an important model system for discovering basic aspects of cell biology. This is especially true for products of early region three (E3), which have provided the foundation for understanding many new mechanisms regulating intracellular trafficking of host cell proteins involved in the host immune response. Cholesterol homeostasis is vital for proper cellular physiology, and disturbances in cholesterol balance are increasingly recognized as important factors in human disease. Despite its central role in numerous aspects of cellular functions, the mechanisms responsible for delivery of dietary cholesterol to the endoplasmic reticulum, where the lipid metabolic and regulatory machinery reside, remain poorly understood. In this review, we describe a novel intracellular pathway for cholesterol trafficking that has been co-opted by an adenovirus E3 gene product. We describe what is known about the molecular regulation of this pathway, how it might benefit viral replication, and its potential involvement in normal cell physiology. Finally, we make a case that adenovirus has co-opted a cellular pathway that may be dysregulated in various human diseases.
    Keywords:  cholesterol; early 3 region; lipid transfer proteins; membrane contact sites
    DOI:  https://doi.org/10.3390/ijms21165808
  37. Neurobiol Dis. 2020 Aug 13. pii: S0969-9961(20)30324-7. [Epub ahead of print]144 105049
    LRRK2 kinase inhibitors reduce alpha-synuclein in human neuronal cell lines with the G2019S mutation.
    Ye Zhao, Shikara Keshiya, Gayathri Perera, Lauren Schramko, Glenda M Halliday, Nicolas Dzamko.
      Kinase activating missense mutations in leucine-rich repeat kinase 2 (LRRK2) predispose to Parkinson's disease. Consequently, there is much interest in delineating LRRK2 biology, both in terms of gaining further insight into disease causes, and also determining whether or not LRRK2 is a potential Parkinson's disease therapeutic target. Indeed, many potent and selective small molecule inhibitors of LRRK2 have been developed and are currently being used for pre-clinical testing in cell and animal models. In the current study, we have obtained fibroblasts from four subjects with the common LRRK2 mutation, G2019S. Fibroblasts were reprogrammed to induced pluripotent stem cells and then to neural stem cells and ultimately neurons. Two clones for each of the human neural cell lines were then chronically treated with and without either of two distinct inhibitors of LRRK2 and effects on toxicity and Parkinson's disease related phenotypes were assessed. Cells with the G2019S mutation had a propensity to accumulate the pathological Parkinson's disease protein α-synuclein. Moreover, α-synuclein accumulation in the G2019S cells was significantly reduced with both LRRK2 inhibitors in seven of the eight cell lines studied. LRRK2 inhibitors also improved the nuclear morphology of G2019S cells and impacted on measures of autophagy and endoplasmic reticulum stress. Lastly, we did not find evidence of inhibitor toxicity under the chronic treatment conditions. These results add to evidence that LRRK2 inhibitors may have utility in the treatment of Parkinson's disease via reducing α-synuclein.
    Keywords:  Kinase inhibitors; LRRK2; Lysosome; Neuron; Parkinson's disease; Pluripotent stem cells; α-synuclein
    DOI:  https://doi.org/10.1016/j.nbd.2020.105049
  38. J Hematol Oncol. 2020 Aug 17. 13(1): 113
    The MAPK and AMPK signalings: interplay and implication in targeted cancer therapy.
    Jimin Yuan, Xiaoduo Dong, Jiajun Yap, Jiancheng Hu.
      Cancer is characterized as a complex disease caused by coordinated alterations of multiple signaling pathways. The Ras/RAF/MEK/ERK (MAPK) signaling is one of the best-defined pathways in cancer biology, and its hyperactivation is responsible for over 40% human cancer cases. To drive carcinogenesis, this signaling promotes cellular overgrowth by turning on proliferative genes, and simultaneously enables cells to overcome metabolic stress by inhibiting AMPK signaling, a key singular node of cellular metabolism. Recent studies have shown that AMPK signaling can also reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components, RAF/KSR family kinases, which affects not only carcinogenesis but also the outcomes of targeted cancer therapies against the MAPK signaling. In this review, we will summarize the current proceedings of how MAPK-AMPK signalings interplay with each other in cancer biology, as well as its implications in clinic cancer treatment with MAPK inhibition and AMPK modulators, and discuss the exploitation of combinatory therapies targeting both MAPK and AMPK as a novel therapeutic intervention.
    Keywords:  AMPK activators; AMPK inhibitors; AMPK signaling; Autophagy; Cellular metabolism; Interplay; RAF/MEK/ERK inhibitors; Ras/RAF/MEK/ERK signaling; Targeted therapy; Tumorigenesis
    DOI:  https://doi.org/10.1186/s13045-020-00949-4
  39. Cancer Discov. 2020 Aug 14.
    LYTACs Target Extracellular and Membrane Proteins for Degradation.
      Lysosome-targeting chimeras (LYTAC) directed extracellular and membrane proteins to lysosomes.
    DOI:  https://doi.org/10.1158/2159-8290.CD-RW2020-119
  40. Cells. 2020 Aug 20. pii: E1928. [Epub ahead of print]9(9):
    TonEBP Promotes β-Cell Survival under ER Stress by Enhancing Autophagy.
    Hyun Je Kang, Eun Jin Yoo, Hwan Hee Lee, Seung Min An, Hyun Park, Whaseon Lee-Kwon, Soo Youn Choi, Hyug Moo Kwon.
      The endoplasmic reticulum (ER) stress response and autophagy are important cellular responses that determine cell fate and whose dysregulation is implicated in the perturbation of homeostasis and diseases. Tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) is a pleiotropic stress protein that mediates both protective and pathological cellular responses. Here, we examined the role of TonEBP in β-cell survival under ER stress. We found that TonEBP increases β-cell survival under ER stress by enhancing autophagy. The level of TonEBP protein increased under ER stress due to a reduction in its degradation via the ubiquitin-proteasome pathway. In response to ER stress, TonEBP increased autophagosome formations and suppressed the accumulation of protein aggregates and β-cell death. The Rel-homology domain of TonEBP interacted with FIP200, which is essential for the initiation of autophagy, and was required for autophagy and cell survival upon exposure to ER stress. Mice in which TonEBP was specifically deleted in pancreatic endocrine progenitor cells exhibited defective glucose homeostasis and a loss of islet mass. Taken together, these findings demonstrate that TonEBP protects against ER stress-induced β-cell death by enhancing autophagy.
    Keywords:  FIP200; NFAT5; UPR; autophagy initiation; islet; unfolded protein response
    DOI:  https://doi.org/10.3390/cells9091928
  41. Cells. 2020 Aug 15. pii: E1904. [Epub ahead of print]9(8):
    Oncology Therapeutics Targeting the Metabolism of Amino Acids.
    Nefertiti Muhammad, Hyun Min Lee, Jiyeon Kim.
      Amino acid metabolism promotes cancer cell proliferation and survival by supporting building block synthesis, producing reducing agents to mitigate oxidative stress, and generating immunosuppressive metabolites for immune evasion. Malignant cells rewire amino acid metabolism to maximize their access to nutrients. Amino acid transporter expression is upregulated to acquire amino acids from the extracellular environment. Under nutrient depleted conditions, macropinocytosis can be activated where proteins from the extracellular environment are engulfed and degraded into the constituent amino acids. The demand for non-essential amino acids (NEAAs) can be met through de novo synthesis pathways. Cancer cells can alter various signaling pathways to boost amino acid usage for the generation of nucleotides, reactive oxygen species (ROS) scavenging molecules, and oncometabolites. The importance of amino acid metabolism in cancer proliferation makes it a potential target for therapeutic intervention, including via small molecules and antibodies. In this review, we will delineate the targets related to amino acid metabolism and promising therapeutic approaches.
    Keywords:  amino acids; cancer metabolism; oncogenic therapeutics
    DOI:  https://doi.org/10.3390/cells9081904
  42. Int J Mol Sci. 2020 Aug 12. pii: E5784. [Epub ahead of print]21(16):
    Predicting the Development of Anti-Drug Antibodies against Recombinant alpha-Galactosidase A in Male Patients with Classical Fabry Disease.
    Sanne J van der Veen, Wytze J Vlietstra, Laura van Dussen, André B P van Kuilenburg, Marcel G W Dijkgraaf, Malte Lenders, Eva Brand, Christoph Wanner, Derralynn Hughes, Perry M Elliott, Carla E M Hollak, Mirjam Langeveld.
      Fabry Disease (FD) is a rare, X-linked, lysosomal storage disease that mainly causes renal, cardiac and cerebral complications. Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A is available, but approximately 50% of male patients with classical FD develop inhibiting anti-drug antibodies (iADAs) that lead to reduced biochemical responses and an accelerated loss of renal function. Once immunization has occurred, iADAs tend to persist and tolerization is hard to achieve. Here we developed a pre-treatment prediction model for iADA development in FD using existing data from 120 classical male FD patients from three European centers, treated with ERT. We found that nonsense and frameshift mutations in the α-galactosidase A gene (p = 0.05), higher plasma lysoGb3 at baseline (p < 0.001) and agalsidase beta as first treatment (p = 0.006) were significantly associated with iADA development. Prediction performance of a Random Forest model, using multiple variables (AUC-ROC: 0.77) was compared to a logistic regression (LR) model using the three significantly associated variables (AUC-ROC: 0.77). The LR model can be used to determine iADA risk in individual FD patients prior to treatment initiation. This helps to determine in which patients adjusted treatment and/or immunomodulatory regimes may be considered to minimize iADA development risk.
    Keywords:  Fabry disease; anti-drug antibodies; enzyme replacement therapy; prediction model
    DOI:  https://doi.org/10.3390/ijms21165784
  43. J Cell Sci. 2020 Aug 14. pii: jcs.245555. [Epub ahead of print]
    Amino acid homeostatic control by TORC1 in Saccharomyces cerevisiae under high hydrostatic pressure.
    Satoshi Uemura, Takahiro Mochizuki, Kengo Amemiya, Goyu Kurosaka, Miho Yazawa, Keiko Nakamoto, Yu Ishikawa, Shingo Izawa, Fumiyoshi Abe.
      Mechanical stresses including high hydrostatic pressure elicit diverse physiological effects on organisms. Gtr1/Gtr2 and Ego1/Ego3, central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25 MPa stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased the glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ, and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1, and PIB2 with GLN3 These results suggested that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC-TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrated the regulatory circuit that maintained the intracellular amino acid homeostasis and suggested the critical roles the EGOC-TORC1 and Pib2-TORC1 complexes played in the growth of yeast under high hydrostatic pressure.
    Keywords:  EGO complex; Glutamine; Gtr1/Gtr2; High hydrostatic pressure; Pib2; Polysome profile; TORC1
    DOI:  https://doi.org/10.1242/jcs.245555
  44. J Pediatr Endocrinol Metab. 2020 Aug 19. pii: /j/jpem.ahead-of-print/jpem-2020-0056/jpem-2020-0056.xml. [Epub ahead of print]
    Analysis of the alpha galactosidase gene: mutation profile and description of two novel mutations with extensive literature review in Turkish population.
    Hüseyin Onay, Hilmi Bolat, Gonca Kılıç Yıldırım, Engin Köse, Sema Kalkan Uçar, Semih Aşıkovalı, Ferda Özkınay, Mahmut Çoker.
      Objectives Fabry disease (FD, OMIM #301500) is a rare and progressive X-linked lysosomal storage disorder. FD is caused by mutations in the GLA gene on chromosome Xq22. Methods In this article, we aimed to present the largest sample of GLA mutation spectrum including common and novel variants in Turkish population. GLA gene sequence analysis was performed on the subjects who applied to the department of medical genetics with the preliminary diagnosis of FD between 2013 and 2018. Results We detected 22 different mutations as two novel [(p.F69S(c.206T>C), p.P205A (c.613C>G)] and 20 previously reported GLA mutations in 47 individuals from 22 unrelated families. These mutations included 14 missense mutations, four nonsense mutations, two small deletions, one small deletion/insertion and one small insertion. Major clinical findings of the female case with p.F69S(c.206T>C) mutation were cornea verticillata, acroparesthesia, angiokeratoma, psychiatric and gastrointestinal symptoms. Other novel mutation (p.P205A [c.613C>G]) was carried by a male case presenting gastrointestinal symptoms. Conclusions We described clinical findings of two cases that had novel mutations to provide more insight in genotype-phenotype correlation. We presented the largest mutation spectrum in Turkish population and reviewed previous mutations in this article.
    Keywords:   GLA ; Fabry disease; alpha-galactosidase
    DOI:  https://doi.org/10.1515/jpem-2020-0056
  45. Am J Physiol Gastrointest Liver Physiol. 2020 Aug 19.
    An overview of deregulated lipid metabolism in nonalcoholic fatty liver disease with special focus on lysosomal acid lipase.
    Simone Carotti, Katia Aquilano, Francesco Valentini, Sergio Ruggiero, Francesca Alletto, Sergio Morini, Antonio Picardi, Raffaele Antonelli-Incalzi, Daniele Lettieri-Barbato, Umberto Vespasiani-Gentilucci.
      Obesity and type 2 diabetes are frequently complicated by excess fat accumulation in the liver, which is known as nonalcoholic fatty liver disease (NAFLD). In this context, liver steatosis develops as a result of the deregulation of pathways controlling de novo lipogenesis and fat catabolism. Recent evidences suggest the clinical relevance of a reduction in the activity of lysosomal acid lipase (LAL), which is a key enzyme for intracellular fat disposal, in patients with NAFLD. In this review, we provided a comprehensive overview of the critical steps in hepatic fat metabolism and alterations in these pathways in NAFLD, with a special focus on lipophagy and LAL activity. During NAFLD, hepatic fat metabolism is impaired at several levels, which is significantly contributed to by impaired lipophagy, in which reduced LAL activity may play an important role. For further research and intervention in NAFLD, targeting LAL activity may provide interesting perspectives.
    Keywords:  Lipid Droplet; Lipogenesis; Lipophagy; Lysosomal acid lipase; Non-alcoholic fatty liver disease
    DOI:  https://doi.org/10.1152/ajpgi.00049.2020
  46. Anticancer Agents Med Chem. 2020 Aug 19.
    N-Terminal-Dependent Protein Degradation and Targeting Cancer Cells.
    Mohamed A Eldeeb.
      Intracellular protein degradation is mediated selectively by the Ubiquitin Proteasome System (UPS) and autophagic-lysosomal system in mammalian cells. Many cellular and physiological processes, such as cell division, cell differentiation, and cellular demise. are fine-tuned via the UPS-mediated protein degradation. Notably, impairment of UPS contributes to human disorders including cancer and neurodegeneration. The proteasome-dependent N-degron pathways mediate the degradation of proteins through their destabilizing amino-terminal residues. Recent advances unveiled that targeting N-degron proteolytic pathways can aid in sensitizing some cancer cells to chemotherapeutic agents. Furthermore, interestingly, exploiting the N-degron feature, the simplest degradation signal in mammals, and fusing it to a ligand specific for Estrogen-Related Receptor alpha (ERRa) has demonstrated its utility in ERRa knockdown, via Nterminal dependent degradation, and also its efficiency in the inhibition of growth of breast cancer cells. These recent advances uncover the therapeutic implications of targeting and exploiting N-degron proteolytic pathways to curb growth and migration of cancer cells.
    Keywords:  Cancer cell death; N-degron; N-end rule; PROTACS. ; apoptosis; proteasome; protein degradation; proteolysis; ubiquitin
    DOI:  https://doi.org/10.2174/1871520620666200819112632
  47. Int J Mol Sci. 2020 Aug 18. pii: E5924. [Epub ahead of print]21(16):
    Osteoclasts' Ability to Generate Trenches Rather Than Pits Depends on High Levels of Active Cathepsin K and Efficient Clearance of Resorption Products.
    Xenia G Borggaard, Dinisha C Pirapaharan, Jean-Marie Delaissé, Kent Søe.
      Until recently, it was well-accepted that osteoclasts resorb bone according to the resorption cycle model. This model is based on the assumption that osteoclasts are immobile during bone erosion, allowing the actin ring to be firmly attached and thereby provide an effective seal encircling the resorptive compartment. However, through time-lapse, it was recently documented that osteoclasts making elongated resorption cavities and trenches move across the bone surface while efficiently resorbing bone. However, it was also shown that osteoclasts making rounded cavities and pits indeed resorb bone while they are immobile. Only little is known about what distinguishes these two different resorption modes. This is of both basic and clinical interest because these resorption modes are differently sensitive to drugs and are affected by the gender as well as age of the donor. In the present manuscript we show that: 1. levels of active cathepsin K determine the switch from pit to trench mode; 2. pit and trench mode depend on clathrin-mediated endocytosis; and 3. a mechanism integrating release of resorption products and membrane/integrin recycling is required for prolongation of trench mode. Our study therefore contributes to an improved understanding of the molecular and cellular determinants for the two osteoclastic bone resorption modes.
    Keywords:  bone resorption; cathepsin K; chloroquine; clathrin; functional secretory domain; lysosome; osteoclast; pit; transcytosis; trench
    DOI:  https://doi.org/10.3390/ijms21165924
  48. Proc Natl Acad Sci U S A. 2020 Aug 19. pii: 202006997. [Epub ahead of print]
    Decoding three distinct states of the Syntaxin17 SNARE motif in mediating autophagosome-lysosome fusion.
    Ying Li, Xiaofang Cheng, Miao Li, Yingli Wang, Tao Fu, Zixuan Zhou, Yaru Wang, Xinyu Gong, Xiaolong Xu, Jianping Liu, Lifeng Pan.
      Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.
    Keywords:  GABARAP; SNARE; Syntaxin17; autophagosome–lysosome fusion; autophagy
    DOI:  https://doi.org/10.1073/pnas.2006997117
  49. Nature. 2020 Aug 19.
    Age-induced accumulation of methylmalonic acid promotes tumour progression.
    Ana P Gomes, Didem Ilter, Vivien Low, Jennifer E Endress, Juan Fernández-García, Adam Rosenzweig, Tanya Schild, Dorien Broekaert, Adnan Ahmed, Melanie Planque, Ilaria Elia, Julie Han, Charles Kinzig, Edouard Mullarky, Anders P Mutvei, John Asara, Rafael de Cabo, Lewis C Cantley, Noah Dephoure, Sarah-Maria Fendt, John Blenis.
      The risk of cancer and associated mortality increases substantially in humans from the age of 65 years onwards1-6. Nonetheless, our understanding of the complex relationship between age and cancer is still in its infancy2,3,7,8. For decades, this link has largely been attributed to increased exposure time to mutagens in older individuals. However, this view does not account for the established role of diet, exercise and small molecules that target the pace of metabolic ageing9-12. Here we show that metabolic alterations that occur with age can produce a systemic environment that favours the progression and aggressiveness of tumours. Specifically, we show that methylmalonic acid (MMA), a by-product of propionate metabolism, is upregulated in the serum of older people and functions as a mediator of tumour progression. We traced this to the ability of MMA to induce SOX4 expression and consequently to elicit transcriptional reprogramming that can endow cancer cells with aggressive properties. Thus, the accumulation of MMA represents a link between ageing and cancer progression, suggesting that MMA is a promising therapeutic target for advanced carcinomas.
    DOI:  https://doi.org/10.1038/s41586-020-2630-0
  50. Biochem Biophys Res Commun. 2020 Aug 17. pii: S0006-291X(20)31557-6. [Epub ahead of print]
    Rictor promotes tumor progression of rapamycin-insensitive triple-negative breast cancer cells.
    Risayo Watanabe, Mamiko Miyata, Chitose Oneyama.
      Triple-negative breast cancer (TNBC), characterized by decreased expression of hormone receptors and human epidermal growth factor type 2 receptor, has poor prognosis and lacks effective therapeutics. Recently, the mTOR inhibitor rapamycin and its analogs have attracted growing interests and evaluated as therapeutic agents against TNBC, in which the PI3K/AKT/mTOR pathway is often activated. However, some TNBCs are less sensitive to these drugs. In this study, we found that the sensitivity of TNBC cells to rapamycin was highly dependent on the expression level of rapamycin-insensitive companion of mTOR (Rictor), a key component of the mTOR complex 2. Repression of the Rictor expression strongly suppressed the growth of rapamycin-insensitive tumor cells. Furthermore, we showed that the suppression of Rictor expression was also effective in rapamycin-insensitive cells that had acquired resistance to mTOR kinase inhibitors. These findings indicate that Rictor can be a predictive marker for the use of rapamycin analogs in TNBC and highlight the need to develop therapeutics targeting Rictor in the treatment of TNBC.
    Keywords:  Rapamycin; Rictor; Triple-negative breast cancer
    DOI:  https://doi.org/10.1016/j.bbrc.2020.08.012
  51. Biochem Biophys Res Commun. 2020 Sep 03. pii: S0006-291X(20)31170-0. [Epub ahead of print]529(4): 1025-1032
    BAP1 regulates AMPK-mTOR signalling pathway through deubiquitinating and stabilizing tumour-suppressor LKB1.
    Cong Yang, Hongyu Ding, Yang Yang, Long Yang, Yun Yang, Meimiao Fang, Jin Ren, Ronggui Hu, Chengcheng Wang, Wujun Geng.
      Liver kinase B1 (LKB1), a tumour suppressor, participates in many cellular processes, including cell survival, growth, apoptosis, transformation, and metabolism. Upon performing yeast two-hybrid screening, co-immunoprecipitation, and GST pull-down, we identified that BRCA1-associated protein 1 (BAP1), a deubiquitinase, interacts with LKB1. Immunoblotting was performed to examine the effect of BAP1 on the activation of 5' AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), downstream of LKB1. The relationship between BAP1 deficiency and cancer cell proliferation was examined using cell survival assay and soft agar assay. qRT-PCR and oil red O staining were performed to evaluate lipid synthesis. Our findings reveal that BAP1 deubiquitinates LKB1, inhibits its degradation, and stabilises it, thereby affecting AMPK activation and downstream mTOR activity. BAP1 deficiency may enhance cellular proliferation as well as lipid synthesis.
    Keywords:  AMPK; BAP1; Deubiquitination; LKB1; mTOR
    DOI:  https://doi.org/10.1016/j.bbrc.2020.05.223
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