bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒09‒27
forty-two papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing


  1. Biochim Biophys Acta Mol Basis Dis. 2020 Sep 17. pii: S0925-4439(20)30318-5. [Epub ahead of print] 165970
    Krishan S, Sahni S, Richardson DR.
      Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and its analogues are potent anti-cancer agents through their ability to target lysosomes. Considering this, it was important to understand the mechanisms involved in the Dp44mT-mediated induction of autophagy and the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) as a critical autophagic regulator. As such, this investigation examined AMPK's role in the regulation of the transcription factor EB (TFEB), which transcribes genes involved in autophagy and lysosome biosynthesis. For the first time, this study demonstrated that Dp44mT induces translocation of TFEB to the nucleus. Furthermore, Dp44mT-mediated nuclear translocation of TFEB was AMPK-dependent. Considering that: (1) the mammalian target of rapamycin complex 1 (mTORC1) plays an important role in the regulation of TFEB; and (2) that AMPK is a known regulator of mTORC1, this study also elucidated the mechanisms through which Dp44mT regulates nuclear translocation of TFEB via AMPK. Silencing AMPK led to increased mTOR phosphorylation, that activates mTORC1. Since Dp44mT inhibits mTORC1 in an AMPK-dependent manner through raptor phosphorylation, Dp44mT is demonstrated to regulate TFEB translocation through dual mechanisms: AMPK activation, which inhibits mTOR, and inhibition of mTORC1 via phosphorylation of raptor. Collectively, Dp44mT-mediated activation of AMPK plays a crucial role in lysosomal biogenesis and TFEB function. As Dp44mT potently chelates copper and iron that are crucial for tumor growth, these studies provide insight into the regulatory mechanisms involved in intracellular clearance and energy metabolism that occur upon alterations in metal ion homeostasis.
    Keywords:  AMPK; Anti-cancer agent; Dp44mT; Molecular pharmacology; Molecular target; pAMPK
    DOI:  https://doi.org/10.1016/j.bbadis.2020.165970
  2. Biomolecules. 2020 Sep 18. pii: E1339. [Epub ahead of print]10(9):
    Meena NK, Raben N.
      Pompe disease, also known as glycogen storage disease type II, is caused by the lack or deficiency of a single enzyme, lysosomal acid alpha-glucosidase, leading to severe cardiac and skeletal muscle myopathy due to progressive accumulation of glycogen. The discovery that acid alpha-glucosidase resides in the lysosome gave rise to the concept of lysosomal storage diseases, and Pompe disease became the first among many monogenic diseases caused by loss of lysosomal enzyme activities. The only disease-specific treatment available for Pompe disease patients is enzyme replacement therapy (ERT) which aims to halt the natural course of the illness. Both the success and limitations of ERT provided novel insights in the pathophysiology of the disease and motivated the scientific community to develop the next generation of therapies that have already progressed to the clinic.
    Keywords:  Pompe disease; autophagy; enzyme replacement therapy; gene therapy; lysosomal targeting; lysosome; muscle; satellite cells
    DOI:  https://doi.org/10.3390/biom10091339
  3. Int J Mol Sci. 2020 Sep 17. pii: E6812. [Epub ahead of print]21(18):
    Khan SA, Tomatsu SC.
      Mucolipidosis II and III (ML II/III) are caused by a deficiency of uridine-diphosphate N-acetylglucosamine: lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. The process is performed by a sequential two-step process: first, GlcNAc-1-phosphotransferase catalyzes the transfer of GlcNAc-1-phosphate to the selected mannose residues on lysosomal enzymes in the cis-Golgi network. The second step removes GlcNAc from lysosomal enzymes by N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (uncovering enzyme) and exposes the mannose 6-phosphate (M6P) residues in the trans-Golgi network, in which the enzymes are targeted to the lysosomes by M6Preceptors. A deficiency of GlcNAc-1-phosphotransferase causes the hypersecretion of lysosomal enzymes out of cells, resulting in a shortage of multiple lysosomal enzymes within lysosomes. Due to a lack of GlcNAc-1-phosphotransferase, the accumulation of cholesterol, phospholipids, glycosaminoglycans (GAGs), and other undegraded substrates occurs in the lysosomes. Clinically, ML II and ML III exhibit quite similar manifestations to mucopolysaccharidoses (MPSs), including specific skeletal deformities known as dysostosis multiplex and gingival hyperplasia. The life expectancy is less than 10 years in the severe type, and there is no definitive treatment for this disease. In this review, we have described the updated diagnosis and therapy on ML II/III.
    Keywords:  I-cell disease; glycosaminoglycans; inclusion body; lysosomal storage disorders; lysosome enzyme transport; mannose 6-phosphate
    DOI:  https://doi.org/10.3390/ijms21186812
  4. Autophagy. 2020 Sep 24.
    Peintner L, Venkatraman A, Waeldin A, Hofherr A, Busch T, Voronov A, Viau A, Kuehn EW, Köttgen M, Borner C.
      Mutations in the PKD1 gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used Pkd1-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for Pkd1, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional pkd1fl/fl;Pax8rtTA;TetOCre+ knockout mice to show that the lack of Pkd1 caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. Pkd1-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in pkd1 knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux.
    Keywords:   Pkd1 ; LAMPs; TFEB; autophagy; calpains; cathepsins; lysosomes; multivesicular bodies; polycystic kidney disease; polycystin-1
    DOI:  https://doi.org/10.1080/15548627.2020.1826716
  5. Sci Adv. 2020 Sep;pii: eabb0205. [Epub ahead of print]6(39):
    Gambardella G, Staiano L, Moretti MN, De Cegli R, Fagnocchi L, Di Tullio G, Polletti S, Braccia C, Armirotti A, Zippo A, Ballabio A, De Matteis MA, di Bernardo D.
      Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.
    DOI:  https://doi.org/10.1126/sciadv.abb0205
  6. Autophagy. 2020 Sep 22. 1-21
    Carosi JM, Hein LK, van den Hurk M, Adams R, Milky B, Singh S, Bardy C, Denton D, Kumar S, Sargeant TJ.
      The macroautophagy/autophagy-lysosome axis enables the clearance and degradation of cytoplasmic components including protein aggregates, damaged organelles and invading pathogens. Protein aggregation and lysosomal system dysfunction in the brain are common features of several late-onset neurological disorders including Alzheimer disease. Spatial overlap between depletion of the endosomal-sorting complex retromer and MAPT/tau aggregation in the brain have been previously reported. However, whether retromer dysfunction plays a direct role in mediating MAPT aggregation remains unclear. Here, we demonstrate that the autophagy-lysosome axis is the primary mode for the clearance of aggregated species of MAPT using both chemical and genetic approaches in cell models of amyloid MAPT aggregation. We show that depletion of the central retromer component VPS35 causes a block in the resolution of autophagy. We establish that this defect underlies marked accumulation of cytoplasmic MAPT aggregates upon VPS35 depletion, and that VPS35 overexpression has the opposite effect. This work illustrates how retromer complex integrity regulates the autophagy-lysosome axis to suppress MAPT aggregation and spread.
    Keywords:  Amyloid; VPS35; autophagy; lysosome; protein aggregation; tauopathy
    DOI:  https://doi.org/10.1080/15548627.2020.1821545
  7. Int J Mol Sci. 2020 Sep 19. pii: E6881. [Epub ahead of print]21(18):
    Ryckman AE, Brockhausen I, Walia JS.
      Glycosphingolipids (GSLs) are a specialized class of membrane lipids composed of a ceramide backbone and a carbohydrate-rich head group. GSLs populate lipid rafts of the cell membrane of eukaryotic cells, and serve important cellular functions including control of cell-cell signaling, signal transduction and cell recognition. Of the hundreds of unique GSL structures, anionic gangliosides are the most heavily implicated in the pathogenesis of lysosomal storage diseases (LSDs) such as Tay-Sachs and Sandhoff disease. Each LSD is characterized by the accumulation of GSLs in the lysosomes of neurons, which negatively interact with other intracellular molecules to culminate in cell death. In this review, we summarize the biosynthesis and degradation pathways of GSLs, discuss how aberrant GSL metabolism contributes to key features of LSD pathophysiology, draw parallels between LSDs and neurodegenerative proteinopathies such as Alzheimer's and Parkinson's disease and lastly, discuss possible therapies for patients.
    Keywords:  biosynthesis; degradation of glycoconjugates; glycosphingolipids; glycosyl hydrolases; lysosomal storage diseases; pathophysiology
    DOI:  https://doi.org/10.3390/ijms21186881
  8. J Neuroinflammation. 2020 Sep 20. 17(1): 277
    Demir SA, Timur ZK, Ateş N, Martínez LA, Seyrantepe V.
      BACKGROUND: Tay-Sachs disease (TSD), a type of GM2-gangliosidosis, is a progressive neurodegenerative lysosomal storage disorder caused by mutations in the α subunit of the lysosomal β-hexosaminidase enzyme. This disease is characterized by excessive accumulation of GM2 ganglioside, predominantly in the central nervous system. Although Tay-Sachs patients appear normal at birth, the progressive accumulation of undegraded GM2 gangliosides in neurons leads to death. Recently, an early onset Tay-Sachs disease mouse model, with genotype Hexa-/-Neu3-/-, was generated. Progressive accumulation of GM2 led to premature death of the double KO mice. Importantly, this double-deficient mouse model displays typical features of Tay-Sachs patients, such as cytoplasmic vacuolization of nerve cells, deterioration of Purkinje cells, neuronal death, deceleration in movement, ataxia, and tremors. GM2-gangliosidosis is characterized by acute neurodegeneration preceded by activated microglia expansion, macrophage, and astrocyte activation, along with the production of inflammatory mediators. However, the mechanism of disease progression in Hexa-/-Neu3-/- mice, relevant to neuroinflammation is poorly understood.METHOD: In this study, we investigated the onset and progression of neuroinflammatory changes in the cortex, cerebellum, and retina of Hexa-/-Neu3-/- mice and control littermates by using a combination of molecular genetics and immunochemical procedures.
    RESULTS: We found elevated levels of pro-inflammatory cytokine and chemokine transcripts, such as Ccl2, Ccl3, Ccl4, and Cxcl10 and also extensive microglial and astrocyte activation and proliferation, accompanied by peripheral blood mononuclear cell infiltration in the vicinity of neurons and oligodendrocytes. Behavioral tests demonstrated a high level of anxiety, and age-dependent loss in both spatial learning and fear memory in Hexa-/-Neu3-/- mice compared with that in the controls.
    CONCLUSION: Altogether, our data suggest that Hexa-/-Neu3-/- mice display a phenotype similar to Tay-Sachs patients suffering from chronic neuroinflammation triggered by GM2 accumulation. Furthermore, our work contributes to better understanding of the neuropathology in a mouse model of early onset Tay-Sachs disease.
    Keywords:  Behavior; GM2; Mouse model; Neuroinflammation; Tay-Sachs disease
    DOI:  https://doi.org/10.1186/s12974-020-01947-6
  9. J Exp Clin Cancer Res. 2020 Sep 22. 39(1): 197
    Yang Y, Wang Q, Song D, Zen R, Zhang L, Wang Y, Yang H, Zhang D, Jia J, Zhang J, Wang J.
      BACKGROUND: Autophagy is an intracellular process through which intracellular components are recycled in response to nutrient or growth factor deficiency to maintain homeostasis. We identified the peptide autophagy-related cancer-suppressing peptide (ARCSP), a potential antitumor peptide that disrupts intracellular homeostasis by blocking autophagic flux and causes cytotoxic death.METHODS: The proliferative ability of ARCSP-treated cervical cancer cells was examined by the CCK8, EdU, and colony formation assays. The TUNEL assay was used to detect apoptosis. Mitochondrial function was evaluated based on the mitochondrial membrane potential. Autophagic flux was detected by immunofluorescence and confocal microscopy. The autophagy-related proteins AMPK, Raptor, mTOR, p62, LC3B, atg7, Rab7, LAMP1, LAMP2, and cathepsin D were detected by Immunoblotting. The antitumor effect of ARCSP was explored in vivo by establishing a transplant tumor model in nude mice.
    RESULTS: The results demonstrated that ARCSP induced cell death and inhibited proliferation. ARCSP induced AMPK/mTOR activation, resulting in the accumulation of the proteins LC3B, p62 and Atg7. ARCSP also blocked autophagosome-lysosome fusion by inhibiting endosomal maturation and increasing the lysosomal pH. The accumulation of nonfused autophagosomes exacerbated cytotoxic death, whereas knocking down Atg7 reversed the cytotoxic death induced by ARCSP. ARCSP-treated cells exhibited increased cytotoxic death after cotreatment with an autophagy inhibitor (Chloroquine CQ). Furthermore, the tumors of ARCSP-treated nude mice were significantly smaller than those of untreated mice.
    CONCLUSIONS: Our findings demonstrate that ARCSP, a novel lethal nonfused autophagosome inducer, might cause mitochondrial dysfunction and autophagy-related cytotoxic death and is thus a prospective agent for cancer therapy.
    Keywords:  ARCSP; Autophagic flux; Autophagy-related cytotoxic death; Cervical cancer; Nonfused autophagosome
    DOI:  https://doi.org/10.1186/s13046-020-01701-z
  10. Exp Physiol. 2020 Sep 23.
    Hodson N, Dent JR, Song Z, O'Leary MF, Nicholson T, Jones SW, Murray JT, Jeromson S, Hamilton DL, Breen L, Philp A.
      NEW FINDINGS: What is the central question of the study? Is Vps34 a nutrient-sensitive activator of mTORC1 in human skeletal muscle? What is the main finding and its importance? We show that altering nutrient availability, via protein-carbohydrate feeding, does not increase Vps34 kinase activity in human skeletal muscle. Instead, feeding increased Vps34-mTORC1 co-localization in parallel to increased mTORC1 activity. These findings may have important implications in the understanding nutrient-induced mTORC1 activation in skeletal muscle via interaction with Vps34.ABSTRACT: The Class III PI3Kinase, Vps34, has recently been proposed as a nutrient sensor, essential for activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). We therefore investigated the effects of increasing nutrient availability through protein-carbohydrate (PRO-CHO) feeding on Vps34 kinase activity and cellular localization in human skeletal muscle. Eight young, healthy males (21 ± 0.5 yrs, 77.7 ± 9.9 kg, 25.9 ± 2.7 kg/m2 , mean ± SD) ingested a PRO-CHO beverage containing 20/44/1 g PRO/CHO/FAT respectively, with skeletal muscle biopsies obtained at baseline and 1 h and 3 h post-feeding. PRO-CHO feeding did not alter Vps34 kinase activity, but did stimulate Vps34 translocation toward the cell periphery (PRE (mean ± SD) - 0.273 ± 0.040, 1 h - 0.348 ± 0.061, Pearson's Coefficient (r)) where it co-localized with mTOR (PRE - 0.312 ± 0.040, 1 h - 0.348 ± 0.069, Pearson's Coefficient (r)). These alterations occurred in parallel to an increase in S6K1 kinase activity (941 ± 466% of PRE at 1 h post-feeding). Subsequent in vitro experiments in C2C12 and human primary myotubes displayed no effect of the Vps34-specific inhibitor SAR405 on mTORC1 signalling responses to elevated nutrient availability. Therefore, in summary, PRO-CHO ingestion does not increase Vps34 activity in human skeletal muscle, whilst pharmacological inhibition of Vps34 does not prevent nutrient stimulation of mTORC1 in vitro. However, PRO-CHO ingestion promotes Vps34 translocation to the cell periphery, enabling Vps34 to associate with mTOR. Therefore, our data suggests that interaction between Vps34 and mTOR, rather than changes in Vps34 activity per se may be involved in PRO-CHO activation of mTORC1 in human skeletal muscle. This article is protected by copyright. All rights reserved.
    Keywords:  Vps34; lysosome; mTORC1
    DOI:  https://doi.org/10.1113/EP088805
  11. FASEB J. 2020 Sep 22.
    López DM, Kählau L, Jungnickel KEJ, Löw C, Damme M.
      The two lysosomal integral membrane proteins MFSD1 and GLMP form a tight complex that confers protection of both interaction partners against lysosomal proteolysis. We here refined the molecular interaction of the two proteins and found that the luminal domain of GLMP alone, but not its transmembrane domain or its short cytosolic tail, conveys protection and mediates the interaction with MFSD1. Our data support the finding that the interaction is essential for the stabilization of the complex. These results are complemented by the observation that N-glycosylation of GLMP in general, but not the type of N-glycans (high-mannose-type or complex-type) or individual N-glycan chains, are essential for protection. We observed that the interaction of both proteins already starts in the endoplasmic reticulum, and quantitatively depends on each other. Both proteins can affect vice versa their intracellular trafficking to lysosomes in addition to the protection from proteolysis. Finally, we provide evidence that MFSD1 can form homodimers both in vitro and in vivo. Our data refine the complex interplay between an intimate couple of a lysosomal transporter and its accessory subunit.
    Keywords:  GLMP; MFSD1; accessory subunit; lysosomal; transporter
    DOI:  https://doi.org/10.1096/fj.202000912RR
  12. Autophagy. 2020 Sep 22. 1-3
    Su H, Wang X.
      Proteasome inhibition (PSMI) is known to activate macroautophagy (autophagy hereafter), but the underlying mechanisms remain to be fully delineated. Here we discuss our recent work identifying an important PPP3/calcineurin-TFEB-SQSTM1/p62 pathway in mediating activation of autophagy by PSMI, a compensatory process for the heart with proteasome malfunction. Through increasing PPP3/calcineurin activity and inhibiting MTOR signaling, PSMI promotes the dephosphorylation and thereby nuclear translocation of TFEB, resulting in transactivation of genes in the autophagic-lysosomal pathway (ALP) such as Mcoln1 and Sqstm1. We have discovered that SQSTM1 is required for not only induction of autophagy but also cardiac activation of TFEB by PSMI, unveiling a novel feedforward role for SQSTM1 in TFEB activation.
    Keywords:  PPP3/calcineurin; PSMC2; SQSTM1/p62; TFEB; macroautophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1816666
  13. Eur J Med Chem. 2020 Sep 13. pii: S0223-5234(20)30792-3. [Epub ahead of print]208 112820
    Chen Y, Zhou X.
      Mammalian target of rapamycin (mTOR) is a highly conserved Serine/Threonine (Ser/Thr) protein kinase, which belongs to phosphatidylinositol-3-kinase-related kinase (PIKK) protein family. mTOR exists as two types of protein complex: mTORC1 and mTORC2, which act as central controller regulating processes of cell metabolism, growth, proliferation, survival and autophagy. The mTOR inhibitors block mTOR signaling pathway, producing anti-inflammatory, anti-proliferative, autophagy and apoptosis induction effects, thus mTOR inhibitors are mainly used in cancer therapy. At present, mTOR inhibitors are divided into four categories: Antibiotic allosteric mTOR inhibitors (first generation), ATP-competitive mTOR inhibitors (second generation), mTOR/PI3K dual inhibitors (second generation) and other new mTOR inhibitors (third generation). In this article, these four categories of mTOR inhibitors and their structures, properties and some clinical researches will be introduced. Among them, we focus on the structure of mTOR inhibitors and try to analyze the structure-activity relationship. mTOR inhibitors are classified according to their chemical structure and their contents are introduced systematically. Moreover, some natural products that have direct or indirect mTOR inhibitory activities are introduced together. In this article, we analyzed the target, binding mode and structure-activity relationship of each generation of mTOR inhibitors and proposed two hypothetic scaffolds (the inverted-Y-shape scaffold and the C-shape scaffold) for the second generation of mTOR inhibitors. These findings may provide some help or reference for drug designing, drug modification or the future development of mTOR inhibitor.
    Keywords:  Anticancer; Drug design; mTOR; mTOR inhibitor
    DOI:  https://doi.org/10.1016/j.ejmech.2020.112820
  14. Acta Pharm Sin B. 2020 Aug;10(8): 1347-1359
    Chen W, Hu Y, Ju D.
      Gene therapy is rapidly emerging as a powerful therapeutic strategy for a wide range of neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Some early clinical trials have failed to achieve satisfactory therapeutic effects. Efforts to enhance effectiveness are now concentrating on three major fields: identification of new vectors, novel therapeutic targets, and reliable of delivery routes for transgenes. These approaches are being assessed closely in preclinical and clinical trials, which may ultimately provide powerful treatments for patients. Here, we discuss advances and challenges of gene therapy for neurodegenerative disorders, highlighting promising technologies, targets, and future prospects.
    Keywords:  AADC, aromatic-l-amino-acid; AAVs, adeno-associated viruses; AD, Alzheimer's disease; ARSA, arylsulfatase A; ASOs, antisense oligonucleotides; ASPA, aspartoacylase; Adeno-associated viruses; Adv, adenovirus; BBB, blood–brain barrier; BCSFB, blood–cerebrospinal fluid barrier; BRB, blood–retina barrier; Bip, glucose regulated protein 78; CHOP, CCAAT/enhancer binding homologous protein; CLN6, ceroidlipofuscinosis neuronal protein 6; CNS, central nervous system; CSF, cerebrospinal fluid; Central nervous system; Delivery routes; ER, endoplasmic reticulum; FDA, U.S. Food and Drug Administration; GAA, lysosomal acid α-glucosidase; GAD, glutamic acid decarboxylase; GDNF, glial derived neurotrophic factor; Gene therapy; HD, Huntington's disease; HSPGs, heparin sulfate proteoglycans; HTT, mutant huntingtin; IDS, iduronate 2-sulfatase; LVs, retrovirus/lentivirus; Lamp2a, lysosomal-associated membrane protein 2a; NGF, nerve growth factor; Neurodegenerative disorders; PD, Parkinson's disease; PGRN, Progranulin; PINK1, putative kinase 1; PTEN, phosphatase and tensin homolog; RGCs, retinal ganglion cells; RNAi, RNA interference; RPE, retinal pigmented epithelial; SGSH, lysosomal heparan-N-sulfamidase gene; SMN, survival motor neuron; SOD, superoxide dismutase; SUMF, sulfatase-modifying factor; TFEB, transcription factor EB; TPP1, tripeptidyl peptidase 1; TREM2, triggering receptor expressed on myeloid cells 2; UPR, unfolded protein response; ZFPs, zinc finger proteins; mTOR, mammalian target of rapamycin; siRNA, small interfering RNA
    DOI:  https://doi.org/10.1016/j.apsb.2020.01.015
  15. Cancer Metab. 2020 ;8 20
    Tait-Mulder J, Hodge K, Sumpton D, Zanivan S, Vazquez A.
      Background: Mitochondrial serine catabolism to formate induces a metabolic switch to a hypermetabolic state with high rates of glycolysis, purine synthesis and pyrimidine synthesis. While formate is a purine precursor, it is not clear how formate induces pyrimidine synthesis.Methods: Here we combine phospho-proteome and metabolic profiling to determine how formate induces pyrimidine synthesis.
    Results: We discover that formate induces phosphorylation of carbamoyl phosphate synthetase (CAD), which is known to increase CAD enzymatic activity. Mechanistically, formate induces mechanistic target of rapamycin complex 1 (mTORC1) activity as quantified by phosphorylation of its targets S6, 4E-BP1, S6K1 and CAD. Treatment with the allosteric mTORC1 inhibitor rapamycin abrogates CAD phosphorylation and pyrimidine synthesis induced by formate. Furthermore, we show that the formate-dependent induction of mTOR signalling and CAD phosphorylation is dependent on an increase in purine synthesis.
    Conclusions: We conclude that formate activates mTORC1 and induces pyrimidine synthesis via the mTORC1-dependent phosphorylation of CAD.
    DOI:  https://doi.org/10.1186/s40170-020-00228-3
  16. Sci Rep. 2020 Sep 25. 10(1): 15737
    Soliman GA, Schooling CM.
      The mammalian Target of Rapamycin complex 1 (mTORC1) nutrient-sensing pathway is a central regulator of cell growth and metabolism and is dysregulated in diabetes. The eukaryotic translation initiation factor 4E (EIF-4E) protein, a key regulator of gene translation and protein function, is controlled by mTORC1 and EIF-4E Binding Proteins (EIF4EBPs). Both EIF4EBPs and ribosomal protein S6K kinase (RP-S6K) are downstream effectors regulated by mTORC1 but converge to regulate two independent pathways. We investigated whether the risk of type 2 diabetes varied with genetically predicted EIF-4E, EIF-4A, EIF-4G, EIF4EBP, and RP-S6K circulating levels using Mendelian Randomization. We estimated the causal role of EIF-4F complex, EIF4EBP, and S6K in the circulation on type 2 diabetes, based on independent single nucleotide polymorphisms strongly associated (p = 5 × 10-6) with EIF-4E (16 SNPs), EIF-4A (11 SNPs), EIF-4G (6 SNPs), EIF4EBP2 (12 SNPs), and RP-S6K (16 SNPs). The exposure data were obtained from the INTERVAL study. We applied these SNPs for each exposure to publically available genetic associations with diabetes from the DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) case (n = 26,676) and control (n = 132,532) study (mean age 57.4 years). We meta-analyzed SNP-specific Wald-estimates using inverse variance weighting with multiplicative random effects and conducted sensitivity analysis. Mendelian Randomization (MR-Base) R package was used in the analysis. The PhenoScanner curated database was used to identify disease associations with SNP gene variants. EIF-4E is associated with a lowered risk of type 2 diabetes with an odds ratio (OR) 0.94, 95% confidence interval (0.88, 0.99, p = 0.03) with similar estimates from the weighted median and MR-Egger. Similarly, EIF-4A was associated with lower risk of type 2 diabetes with odds ratio (OR) 0.90, 95% confidence interval (0.85, 0.97, p = 0.0003). Sensitivity analysis using MR-Egger and weighed median analysis does not indicate that there is a pleiotropic effect. This unbiased Mendelian Randomization estimate is consistent with a protective causal association of EIF-4E and EIF-4A on type 2 diabetes. EIF-4E and EIF-4A may be targeted for intervention by repurposing existing therapeutics to reduce the risk of type 2 diabetes.
    DOI:  https://doi.org/10.1038/s41598-020-71987-8
  17. Protein Expr Purif. 2020 Sep 16. pii: S1046-5928(20)30343-0. [Epub ahead of print] 105752
    Dwyer B, Hu J, Madduri A, Lundberg D, Miller B, Gill J, Meiyappan M, Pan C, Miller T, Zhang B.
      Alpha galactosidase (a-Gal) is an acidic hydrolase that plays a critical role in hydrolyzing the terminal alpha-galactoyl moiety from glycolipids and glycoproteins. There are over a hundred mutations reported for the GLA gene that encodes a-Gal that result in reduced protein synthesis, protein instability, and reduction in function. The deficiencies of a-Gal can cause Fabry disease, a rare lysosomal storage disorder (LSD) caused by the failure to catabolize alpha-D-galactoyl glycolipid moieties. The current standard of care for Fabry disease is enzyme replacement therapy (ERT) where the purified recombinant form of human a-Gal is given to patients. The manufacture of a-Gal is currently performed utilizing traditional large-scale chromatography processes. Developing an affinity resin for the purification of a-Gal would reduce the complexity of the manufacturing process, reduce costs, and potentially produce a higher quality a-Gal. After the evaluation of many small molecules, a commercially available small molecule biomimetic, N-5-Carboxypentyl-1-deoxygalactonojirimycin (N5C-DGJ), was utilized for the development of a novel small molecule biomimetic affinity resin for a-Gal. Affinity purified a-Gal demonstrated a purity greater than 90%, exhibited expected thermal stability and specific activity. Complementing this affinity purification is the development of an elution buffer system that confers an increased thermal stability to a-Gal. The N5C-DGJ affinity resin tolerated sodium hydroxide sanitization with no loss of binding capacity, making it amenable to large scale purification processes and potential use in manufacturing. This novel method for purifying the challenging a-Gal enzyme can be extended to other enzyme replacement therapies.
    Keywords:  N-5-Carboxypentyl-1-deoxygalactonojirimycin; alpha galactosidase; biomimetic; characterization
    DOI:  https://doi.org/10.1016/j.pep.2020.105752
  18. Eur J Pharmacol. 2020 Sep 16. pii: S0014-2999(20)30654-3. [Epub ahead of print] 173562
    Kong W, YanqingYao , Zhang J, Lu C, Ding Y, Meng Y.
      Mucopolysaccharidosis III (Sanfilippo syndrome, MPS III) is caused by lysosomal enzyme deficiency, which is a rare autosomal recessive hereditary disease. For now, there is no approved treatment for MPS III despite lots of efforts providing new vision of its molecular basis, as well as governments providing regulatory and economic incentives to stimulate the development of specific therapies. Those efforts and incentives attract academic institutions and industry to provide potential therapies for MPS III, including enzyme replacement therapies, substrate reduction therapies, gene and cell therapies, and so on, which were discussed in this paper.
    Keywords:  Enzyme replacement therapy; Gene therapy; Hematopoietic stem cell transplantation; Mucopolysaccharidosis III; Sanfilippo syndrome; Substrate reduction therapy
    DOI:  https://doi.org/10.1016/j.ejphar.2020.173562
  19. Front Oncol. 2020 ;10 1561
    Birkeland ES, Koch LM, Dechant R.
      To adjust cell growth and proliferation to changing environmental conditions or developmental requirements, cells have evolved a remarkable network of signaling cascades that integrates cues from cellular metabolism, growth factor availability and a large variety of stresses. In these networks, cellular information flow is mostly mediated by posttranslational modifications, most notably phosphorylation, or signaling molecules such as GTPases. Yet, a large body of evidence also implicates cytosolic pH (pHc) as a highly conserved cellular signal driving cell growth and proliferation, suggesting that pH-dependent protonation of specific proteins also regulates cellular signaling. In mammalian cells, pHc is regulated by growth factor derived signals and responds to metabolic cues in response to glucose stimulation. Importantly, high pHc has also been identified as a hall mark of cancer, but mechanisms of pH regulation in cancer are only poorly understood. Here, we discuss potential mechanisms of pH regulation with emphasis on metabolic signals regulating pHc by Na+/H+-exchangers. We hypothesize that elevated NHE activity and pHc in cancer are a direct consequence of the metabolic adaptations in tumor cells including enhanced aerobic glycolysis, generally referred to as the Warburg effect. This hypothesis not only provides an explanation for the growth advantage conferred by a switch to aerobic glycolysis beyond providing precursors for accumulation of biomass, but also suggests that treatments targeting pH regulation as a potential anti-cancer therapy may effectively target the result of altered tumor cell metabolism.
    Keywords:  Na+/H+-exchanger; aerobic glycolysis; cytosolic pH; growth and proliferation; metabolism
    DOI:  https://doi.org/10.3389/fonc.2020.01561
  20. Nat Commun. 2020 Sep 25. 11(1): 4865
    Chamoli M, Goyala A, Tabrez SS, Siddiqui AA, Singh A, Antebi A, Lithgow GJ, Watts JL, Mukhopadhyay A.
      The metabolic state of an organism instructs gene expression modalities, leading to changes in complex life history traits, such as longevity. Dietary restriction (DR), which positively affects health and life span across species, leads to metabolic reprogramming that enhances utilisation of fatty acids for energy generation. One direct consequence of this metabolic shift is the upregulation of cytoprotective (CyTP) genes categorized in the Gene Ontology (GO) term of "Xenobiotic Detoxification Program" (XDP). How an organism senses metabolic changes during nutritional stress to alter gene expression programs is less known. Here, using a genetic model of DR, we show that the levels of polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA) and eicosapentaenoic acid (EPA), are increased following DR and these PUFAs are able to activate the CyTP genes. This activation of CyTP genes is mediated by the conserved p38 mitogen-activated protein kinase (p38-MAPK) pathway. Consequently, genes of the PUFA biosynthesis and p38-MAPK pathway are required for multiple paradigms of DR-mediated longevity, suggesting conservation of mechanism. Thus, our study shows that PUFAs and p38-MAPK pathway function downstream of DR to help communicate the metabolic state of an organism to regulate expression of CyTP genes, ensuring extended life span.
    DOI:  https://doi.org/10.1038/s41467-020-18690-4
  21. Front Neurol. 2020 ;11 830
    Zhong J, Jiang F, Yang H, Li J, Cheng J, Zeng Q, Xu Q.
      Krabbe disease (KD), also referred to as globoid cell leukodystrophy, is a rare autosomal recessive lysosomal storage disorder caused by β-galactocerebrosidase (GALC) deficiency. Most patients affected by this disease are infants, and <10% of cases suffer from adult-onset KD. In this study, two Chinese males presented with long-term progressive weakness in their limbs. Magnetic resonance imaging of the brain and spinal cord of these patients revealed lesions with abnormally high signal intensity on T2-weighted (T2W) and T2W fluid-attenuated inversion recovery images. Whole-exome sequencing was performed for both patients, and four GALC mutations were identified. Case 1 carried a novel deletion mutation (p.T633Tfs*2) and a known missense mutation (p.T529M), while case 2 carried a novel missense mutation (p.W355C) and a known missense mutation (p.P154H). Previous literature has rarely reported myelopathy in patients with KD; in this study, we report two cases of adult-onset KD who both experienced myelopathy. We also conducted a literature review of KD and its association with myelopathy. Our findings provide a better understanding of the phenotypic and genotypic profiles associated with adult-onset KD. We recommend that physicians consider KD as a possible diagnosis in cases showing progressive motor dysfunction or gait disorder in association with typical myelopathy.
    Keywords:  Krabbe disease; adult onset; literature review; myelopathy; novel GALC mutations
    DOI:  https://doi.org/10.3389/fneur.2020.00830
  22. Bratisl Lek Listy. 2020 ;121(10): 760-766
    Pakanova Z, Nemcovic M, Ziburova J, Mucha J, Salingova A, Sebova C, Jurickova K, Barath P.
      Inherited metabolic disorders of glycoconjugate metabolism include congenital disorders of glycosylation (CDG) - disorders in biosynthesis of glycoconjugates; and some of the lysosomal storage diseases (LSD) - disorders of their degradation. This review summarizes the brief characteristics of metabolic pathways of synthesis and catabolism of glycoconjugates as well as the latest update of relevant enzymatic defects discovered in population. Every year the number of known subtypes of these disorders dramatically increases as a result of high-throughput analytical infrastructure applied. However, due to the broad spectrum of unspecific clinical symptoms, many patients remain undiagnosed or have wrong diagnosis with ineffective treatment. Thus, disorders of glycoconjugate metabolism should be considered and ruled out in any unexplained syndrome. The collaboration between scientists and physicians plays an important role in the progress of such personalized diagnostics, that is essential mainly for rare diseases (Tab. 2, Fig. 1, Ref. 49). Keywords: congenital disorders of glycosylation, lysosomal storage disorders.
    DOI:  https://doi.org/10.4149/BLL_2020_124
  23. Life Sci. 2020 Sep 22. pii: S0024-3205(20)31237-6. [Epub ahead of print] 118484
    Li X, Du Y, Zhang L, Chen X, He D.
      AIM: Chloride channel 7 (CLC-7), broadly expressed in kidney tissues, affects the lysosome degradation pathway. And redox status impairment contributes to cell apoptosis and activates autophagy flux. This study mainly investigates the role and molecular mechanism of CLC-7 in redox status impairment-induced autophagic flux and apoptosis.MAIN METHODS: When NRK52E cells, rat renal tubular epithelial cells, were exposed to H2O2 treatment, apoptosis, autophagy flux, and CLC-7 expression were detected. Further investigation was done to observe the change of apoptosis and autophagy flux in renal cells under overexpression or knocking down of CLC-7. The lysosomes acidity, lysosome enzyme Cathepsin D activity and phosphorylation of Ampk/mTOR were also examined when CLC-7 was overexpressed or knocked down.
    KEY FINDINGS: Redox status impairment induced apoptosis and autophagy flux in NRK52E cells and upregulated CLC-7. Overexpression of CLC-7 increased lysosome acidity and Cathepsin D activity. In cells with CLC-7 overexpression, we observed a significant increase of autophagy flux and decline of apoptosis, as well as an apparent increase of p-Ampk and decrease of p-mTOR. On the contrary, cells with knocking down CLC-7 led to opposite results.
    SIGNIFICANCES: CLC-7 is essential to maintain and enhance acidity and enzyme activity in lysosome. Through activating autophagy flux, it exerts survival against renal tubular epithelial cell apoptosis induced by redox status impairment. Its function to modulate Ampk/mTOR pathway is the possible reason why CLC-7 can trigger autophagy flux.
    Keywords:  Apoptosis; Autophagy; CLC-7; Redox status impairment; Renal tubular epithelial cell
    DOI:  https://doi.org/10.1016/j.lfs.2020.118484
  24. Mol Biol Cell. 2020 Sep 23. mbcE20030200
    Le L, Escobar IE, Ho T, Lefkovith AJ, Latteri E, Haltaufderhyde KD, Dennis MK, Plowright L, Sviderskaya EV, Bennett DC, Oancea E, Marks MS.
      SLC45A2 encodes a putative transporter expressed primarily in pigment cells. SLC45A2 mutations cause oculocutaneous albinism type IV (OCA4) and polymorphisms are associated with pigmentation variation, but the localization, function, and regulation of SLC45A2 and its variants remain unknown. We show that SLC45A2 localizes to a cohort of mature melanosomes that only partially overlaps with those expressing the chloride channel OCA2. SLC45A2 expressed ectopically in HeLa cells localizes to lysosomes and raises lysosomal pH, suggesting that in melanocytes, SLC45A2 expression, like OCA2 expression, results in the deacidification of maturing melanosomes to support melanin synthesis. Interestingly, OCA2 overexpression compensates for loss of SLC45A2 expression in pigmentation. Analyses of SLC45A2- and OCA2-deficient mouse melanocytes show that SLC45A2 likely functions later during melanosome maturation than OCA2. Moreover, the light skin-associated SLC45A2 allelic F374 variant restores only moderate pigmentation to SLC45A2-deficient melanocytes due to rapid proteasome-dependent degradation resulting in lower protein expression levels in melanosomes than the dark skin-associated allelic variant. Our data suggest that SLC45A2 maintains melanosome neutralization initially orchestrated by transient OCA2 activity to support melanization at late stages of melanosome maturation, and that a common variant imparts reduced activity due to protein instability.
    DOI:  https://doi.org/10.1091/mbc.E20-03-0200
  25. Biochem Biophys Res Commun. 2020 Sep 21. pii: S0006-291X(20)31784-8. [Epub ahead of print]
    Lee SW, Commisso C.
      The interplay between nutrient scarcity and signal transduction circuits is an important aspect of tumorigenesis that regulates many aspects of cancer progression. Glutamine is a critical nutrient for cancer cells, as it contributes to biosynthetic reactions that sustain cancer proliferation and growth. In tumors, because nutrient utilization can often outpace supply, glutamine levels can become limiting and oncogene-mediated metabolic rewiring triggers signaling cascades that support nutrient stress survival. Recently, we identified that in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine depletion can trigger p21-activated kinase (Pak) activation through EGFR signaling as a means to circumvent metabolic stress. Here, we elucidate that glutamine starvation, as well EGF stimulation, can enhance the presence of many different Pak phosphoforms, and that this activation only occurs in a subset of PDAC cells. Pak is a well-established effector of Rac1, and while Rac1 mutant variants can modulate the metabolic induction of Pak phosphorylation, Rac1 inhibition only partially attenuates Pak activation upon glutamine depletion. We decipher that in order to efficiently suppress metabolic activation of Pak, both EGFR and Rac1 signaling must be inhibited. These results provide a mechanistic understanding of how glutamine-regulated signal transduction can control Pak activation in PDAC cells.
    Keywords:  EGFR; Glutamine; Nutrient stress; Pak; Pancreatic; Rac
    DOI:  https://doi.org/10.1016/j.bbrc.2020.09.043
  26. PLoS One. 2020 ;15(9): e0238697
    Woolley SA, Tsimnadis ER, Lenghaus C, Healy PJ, Walker K, Morton A, Khatkar MS, Elliott A, Kaya E, Hoerner C, Priestman DA, Shepherd D, Platt FM, Porebski BT, Willet CE, O'Rourke BA, Tammen I.
      Niemann-Pick type C disease is a lysosomal storage disease affecting primarily the nervous system that results in premature death. Here we present the first report and investigation of Niemann-Pick type C disease in Australian Angus/Angus-cross calves. After a preliminary diagnosis of Niemann-Pick type C, samples from two affected calves and two obligate carriers were analysed using single nucleotide polymorphism genotyping and homozygosity mapping, and NPC1 was considered as a positional candidate gene. A likely causal missense variant on chromosome 24 in the NPC1 gene (NM_174758.2:c.2969C>G) was identified by Sanger sequencing of cDNA. SIFT analysis, protein alignment and protein modelling predicted the variant to be deleterious to protein function. Segregation of the variant with disease was confirmed in two additional affected calves and two obligate carrier dams. Genotyping of 403 animals from the original herd identified an estimated allele frequency of 3.5%. The Niemann-Pick type C phenotype was additionally confirmed via biochemical analysis of Lysotracker Green, cholesterol, sphingosine and glycosphingolipids in fibroblast cell cultures originating from two affected calves. The identification of a novel missense variant for Niemann-Pick type C disease in Angus/Angus-cross cattle will enable improved breeding and management of this disease in at-risk populations. The results from this study offer a unique opportunity to further the knowledge of human Niemann-Pick type C disease through the potential availability of a bovine model of disease.
    DOI:  https://doi.org/10.1371/journal.pone.0238697
  27. Front Oncol. 2020 ;10 1454
    Romano G, Nigita G, Calore F, Saviana M, Le P, Croce CM, Acunzo M, Nana-Sinkam P.
      Lung cancer is the leading cause of cancer mortality worldwide. Increased understanding of the molecular mechanisms of the disease has led to the development of novel therapies and improving outcomes. Recently, extracellular vesicles (EVs) have emerged as vehicles for the transfer of genetic information between tumors and their microenvironment and have been implicated in lung cancer initiation, progression, and response to therapy. However, the mechanisms that drive the biogenesis and selective packaging of EVs remain poorly understood. Rab family guanosine triphosphates (GTPases) and their regulators are important membrane trafficking organizers. In this study, we investigated the role of select Rab GTPases on the regulation of EV release. We found that microRNAs target Rab GTPases to regulate EV release from lung cancer cell lines. In particular, Rab32 is a target of miR-124a, and siRNA and miRNA mediated inhibition of Rab32 leads to impaired EV secretion. The downstream implications for microRNA-based regulation of EV release are currently under investigation.
    Keywords:  Rab27; Rab32; extracellular vesicles; lung cancer; miR-124a
    DOI:  https://doi.org/10.3389/fonc.2020.01454
  28. Cell Res. 2020 Sep 21.
    Wei D, Zhan W, Gao Y, Huang L, Gong R, Wang W, Zhang R, Wu Y, Gao S, Kang T.
      Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.
    DOI:  https://doi.org/10.1038/s41422-020-00409-1
  29. Biochim Biophys Acta Mol Basis Dis. 2020 Sep 17. pii: S0925-4439(20)30319-7. [Epub ahead of print] 165971
    Li B, Wu J, Bao J, Han X, Shen S, Ye X, Dai J, Wu Z, Niu M, He Y, Ni J, Wen L, Wang X, Hu G.
      Acute pancreatitis (AP) is associated with impaired acinar cell autophagic flux, intracellular zymogen activation, cell necrosis and inflammation. Activation of the cholinergic system of vagus nerve has been shown to attenuate AP, but the effect of organ-intrinsic cholinergic system on pancreatitis remains unknown. In this study, we aim to examine the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation within the pancreas during AP. In vivo, AP was induced by caerulein plus LPS or ethanol plus palmitoleic acid in mice. In vitro, pancreatic acini were isolated and subjected to cholecystokinin (CCK) stimulation. Mice or acini were pre-treated with PNU-282987 (selective α7nAChR agonist) or methyllycaconitine citrate salt (selective α7nAChR antagonist). Pancreatitis severity, acinar cell injury, autophagic flux, and transcription factor EB (TFEB) pathway were analyzed. Both caerulein plus LPS in vivo and CCK in vitro led to an up-regulation of α7nAChR, indicating activation of pancreas-intrinsic α7nAChR signaling during AP. PNU-282987 decreased acinar cell injury, trypsinogen activation and pancreatitis severity. Conversely, methyllycaconitine citrate salt increased acinar cell injury and aggravated AP. Moreover, activation of α7nAChR by PNU-282987 promoted autophagic flux as indicated by reduced p62, increased LysoTracker staining and decreased number of autolysosomes with undegraded contents. Furthermore, PNU-282987 treatment significantly increased TFEB activity in pancreatic acinar cells. α7nAChR activation also attenuated pancreatic inflammation and NF-κB activation. Our results showed that activation of α7nAChR protected against experimental pancreatitis through enhancing TFEB-mediated acinar cell autophagy, suggesting that activation of pancreas-intrinsic α7nAChR may serve as an endogenous protective mechanism during AP.
    Keywords:  TFEB; acute pancreatitis; autophagic flux; lysosomal degradation; α7nAChR
    DOI:  https://doi.org/10.1016/j.bbadis.2020.165971
  30. Sci Rep. 2020 Sep 23. 10(1): 15473
    Gerashchenko MV, Nesterchuk MV, Smekalova EM, Paulo JA, Kowalski PS, Akulich KA, Bogorad R, Dmitriev SE, Gygi S, Zatsepin T, Anderson DG, Gladyshev VN, Koteliansky VE.
      Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.
    DOI:  https://doi.org/10.1038/s41598-020-72399-4
  31. Traffic. 2020 Sep 24.
    Waschbüsch D, Khan AR.
      Rab GTPases are master regulators of membrane trafficking in eukaryotic cells. Phosphorylation of Rab GTPases was characterized in the 1990s and there have been intermittent reports of its relevance to Rab functions. Phosphorylation as a regulatory mechanism has gained prominence through the identification of Rabs as physiological substrates of leucine-rich repeat kinase 2 (LRRK2). LRRK2 is a Ser/Thr kinase that is associated with inherited and sporadic forms of Parkinsons's disease. In recent years, numerous kinases and their associated signaling pathways have been identified that lead to phosphorylation of Rabs. These emerging studies suggest that serine/threonine and tyrosine phosphorylation of Rabs may be a widespread and under-appreciated mechanism for controlling their membrane trafficking functions. Here we survey current knowledge of Rab phosphorylation and discuss models for how this post-translational mechanism exerts control of membrane trafficking. This article is protected by copyright. All rights reserved.
    Keywords:  GDP/GTP dissociation inhibitor; GDP/GTP exchange factor; GTPase activating protein; Rab GTPase; Ser/Thr kinase; effector; membrane trafficking; phosphorylation; tyrosine kinase
    DOI:  https://doi.org/10.1111/tra.12765
  32. J Biol Chem. 2020 Sep 23. pii: jbc.RA120.014532. [Epub ahead of print]
    Kaur S, Chen Y, Shenoy SK.
      The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a 7-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels.  Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking.  Using endocytic colocalization and ubiquitination assays we have identified a correlation between the ubiquitination profile and recycling of the GCGR.  Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell-surface.  Glucagon-stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs.  Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant-negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, while a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By downregulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule binding protein (STAMBP) and ubiquitin specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes.  A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated, and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling, and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.
    Keywords:  G protein-coupled receptor (GPCR); deubiquitylation (deubiquitination); endocytosis; glucagon; receptor recycling
    DOI:  https://doi.org/10.1074/jbc.RA120.014532
  33. J Neurochem. 2020 Sep 22.
    Overhoff M, De Bruyckere E, Kononenko NL.
      Multiple aspects of neuronal physiology crucially depend on two cellular pathways, autophagy and endocytosis. During endocytosis, extracellular components either unbound or recognized by membrane-localized receptors (termed "cargo") become internalized into plasma membrane-derived vesicles. These can serve to either recycle the material back to the plasma membrane or send it for degradation to lysosomes. Autophagy also uses lysosomes as a terminal degradation point, although instead of degrading the plasma membrane-derived cargo, autophagy eliminates detrimental cytosolic material and intracellular organelles, which are transported to lysosomes by means of double-membrane vesicles, referred to as autophagosomes. Neurons, like all non-neuronal cells, capitalize on autophagy and endocytosis to communicate with the environment and maintain protein and organelle homeostasis. Additionally, the highly polarized, post-mitotic nature of neurons made them adopt these two pathways for cell-specific functions. These include the maintenance of the synaptic vesicle pool in the presynaptic terminal and the long-distance transport of signaling molecules. Originally discovered independently from each other, it is now clear that autophagy and endocytosis are closely interconnected and share several common participating molecules. Considering the crucial role of autophagy and endocytosis in cell type-specific functions in neurons, it is not surprising that defects in both pathways have been linked to the pathology of numerous neurodegenerative diseases. In this review, we highlight the recent knowledge of the role of endocytosis and autophagy in neurons with a special focus on synaptic physiology and discuss how impairments in genes coding for autophagy and endocytosis proteins can cause neurodegeneration.
    Keywords:  amphisomes; autophagy; endocytosis; neurodegeneration; protein aggregations; synaptic vesicles
    DOI:  https://doi.org/10.1111/jnc.15194
  34. Cell Death Dis. 2020 Sep 24. 11(9): 797
    Liu R, Chen Y, Liu G, Li C, Song Y, Cao Z, Li W, Hu J, Lu C, Liu Y.
      Multidrug resistance (MDR) is the dominant challenge in the failure of chemotherapy in cancers. Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that spreads intracellular signal cascades and regulates a variety of cellular processes. PI3Ks are considered significant causes of chemoresistance in cancer therapy. Protein kinase B (AKT) is also a significant downstream effecter of PI3K signaling, and it modulates several pathways, including inhibition of apoptosis, stimulation of cell growth, and modulation of cellular metabolism. This review highlights the aberrant activation of PI3K/AKT as a key link that modulates MDR. We summarize the regulation of numerous major targets correlated with the PI3K/AKT pathway, which is further related to MDR, including the expression of apoptosis-related protein, ABC transport and glycogen synthase kinase-3 beta (GSK-3β), synergism with nuclear factor kappa beta (NF-κB) and mammalian target of rapamycin (mTOR), and the regulation of glycolysis.
    DOI:  https://doi.org/10.1038/s41419-020-02998-6
  35. Orphanet J Rare Dis. 2020 Sep 23. 15(1): 262
    Di Rocco M, Di Fonzo A, Barbato A, Cappellini MD, Carubbi F, Giona F, Giuffrida G, Linari S, Pession A, Quarta A, Scarpa M, Spada M, Strisciuglio P, Andria G.
      BACKGROUND: How to address the counseling of lifetime risk of developing Parkinson's disease in patients with Gaucher disease and their family members carrying a single variant of the GBA1 gene is not yet clearly defined. In addition, there is no set way of managing Gaucher disease patients, taking into account the possibility that they may show features of Parkinson's disease.METHODS: Starting from an overview on what has recently changed in our knowledge on this issue and grouping the experiences of healthcare providers of Gaucher disease patients, we outline a path of counseling and management of Parkinson's disease risk in Gaucher disease patients and their relatives.
    CONCLUSION: The approach proposed here will help healthcare providers to communicate Parkinson's disease risk to their patients and will reduce the possibility of patients receiving inaccurate information from inadequate sources. Furthermore, this resource will help to empower healthcare providers to identify early signs and/or symptoms of Parkinson's disease and decide when to refer these patients to the neurologist for appropriate specific therapy and follow-up.
    Keywords:  Counseling; Gaucher disease; Management; Risk of Parkinson’s disease
    DOI:  https://doi.org/10.1186/s13023-020-01529-y
  36. Chem Sci. 2020 Jun 21. 11(23): 5855-5865
    Zhang M, Jang H, Nussinov R.
      The search is on for effective specific inhibitors for PI3Kα mutants. PI3Kα, a critical lipid kinase, has two subunits, catalytic and inhibitory. PIK3CA, the gene that encodes the p110α catalytic subunit is a highly mutated protein in cancer. Dysregulation of PI3Kα signalling is commonly associated with tumorigenesis and drug resistance. Despite its vast importance, only recently the FDA approved the first drug (alpelisib by Novartis) for breast cancer. A second (GDC0077), classified as PI3Kα isoform-specific, is undergoing clinical trials. Not surprisingly, these ATP-competitive drugs commonly elicit severe concentration-dependent side effects. Here we briefly review PI3Kα mutations, focus on PI3K drug repertoire and propose new, to-date unexplored PI3Kα therapeutic strategies. These include (1) an allosteric and orthosteric inhibitor combination and (2) taking advantage of allosteric rescue mutations to guide drug discovery.
    DOI:  https://doi.org/10.1039/d0sc01676d
  37. Biochem Biophys Res Commun. 2020 Sep 21. pii: S0006-291X(20)31770-8. [Epub ahead of print]
    Lee SW, Commisso C.
      Nutrient stress driven by glutamine deficiency activates EGFR signaling in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cells. EGFR signaling in the context of glutamine starvation is thought to be instigated by the transcriptional upregulation of EGFR ligands and functions as an adaptation mechanism to allow PDAC cells to maintain metabolic fitness. Having a clear view of the intricate signaling cascades potentiated by the metabolic induction of EGFR is important in understanding how these effector pathways influence cancer progression. In this study, we examined the complex signaling that occurs in PDAC cells when EGFR is activated by glutamine deprivation. We elucidate that the metabolic activation of EGFR is principally mediated by HB-EGF, and that other members of the ErbB receptor tyrosine kinase family are not activated by glutamine starvation. Additionally, we determine that glutamine depletion-driven EGFR signaling is associated with a specific receptor phosphorylation known to participate in a feedback loop, a process that is dependent on Erk. Lastly, we determine that K-Ras is required for glutamine depletion-induced Erk activation, as well as EGFR feedback phosphorylation, but is dispensable for Akt activation. These data provide important insights into the regulation of EGFR signaling in the context of metabolic stresses.
    Keywords:  Akt; EGFR; Erk; Glutamine; Metabolism; Nutrient stress; Ras
    DOI:  https://doi.org/10.1016/j.bbrc.2020.09.029
  38. Mech Ageing Dev. 2020 Sep 19. pii: S0047-6374(20)30154-8. [Epub ahead of print] 111358
    Dakic T, Jevdjovic T, Djordjevic J, Vujovic P.
      It is known that insulin secreted by pancreatic β-cells enters the brain by crossing the blood-brain barrier. However, it was demonstrated that insulin expression occurs in various brain regions as well. Albeit the list of insulin actions in the brain is long and it includes control of energy homeostasis, neuronal survival, maintenance of synaptic plasticity and cognition, not much is known about the adaptive significance of insulin synthesis in brain. We previously reported that short-term fasting promotes insulin expression and subsequent activation of insulin receptor in the rat periventricular nucleus. In order to uncover a physiological importance of the fasting-induced insulin expression in hypothalamus, we analyzed the effect of short-term food deprivation on the expression of several participants of PI3K/AKT/mTOR and Ras/MAPK signaling pathways that are typically activated by this hormone. We found that the hypothalamic content of total and activated IRS1, IRS2, PI3K, and mTOR remained unchanged, but phosphorylated AKT1/2/3 was decreased. The levels of activated ERK1/2 were increased after six-hour fasting. Moreover, activated ERK1/2 was co-expressed with activated insulin receptor in the nucleus arcuatus. Our previously published and current findings suggest that the ERK activation in hypothalamus was at least partially initiated by the centrally produced insulin.
    Keywords:  ERK; fasting; hypothalamus; insulin signaling; rat
    DOI:  https://doi.org/10.1016/j.mad.2020.111358
  39. ACS Appl Mater Interfaces. 2020 Sep 24.
    Liu H, Zhang P, Zhang C, Chen J, Jiang J.
      Exploiting an intelligent fluorescent probe, which can precisely target to lysosome of hepatoma cells and enable accurate molecular imaging, is a key challenge in hepatoma diagnoses. Herein, a single-dye-based polymer nanoprobe (named SPN) with dual targeting and self-calibrating ratiometric characteristics is rationally fabricated via a simple self-assembly strategy for accurate hypochlorous acid (HClO) imaging in lysosome of HepG2 cells. Of note, the covalent incorporation of self-calibrating ratiometric fluorophore (pyrene derivatives) into the core of polymer nanoparticles can not only validly avoid the leakage of fluorophores, but also greatly enhance their brightness. Besides, this polymer nanoprobe (SPN) displays high water dispersibility, ultrafast response (< 1 s), favorable selectivity, outstanding long-term stability (>90 days) and good biocompatibility. Furthermore, thanks to the hepatocyte targeting moiety (galactose) and the interplay of surface charge and size of nanoparticles, the SPN is able to enter into asialoglycoprotein-receptor positive HepG2 cells and further locate at lysosomes, successfully enabling accurate HClO detection in lysosomes of HepG2 cells. This study demonstrates that the versatile SPN can provide more precise dual targeting and accurate molecular imaging.
    DOI:  https://doi.org/10.1021/acsami.0c13857
  40. J Kidney Cancer VHL. 2020 ;7(3): 5-19
    Nair N, Chakraborty R, Mahajan Z, Sharma A, Sethi SK, Raina R.
      Tuberous sclerosis complex (TSC) is a genetic condition caused by a mutation in either the TSC1 or TSC2 gene. Disruption of either of these genes leads to impaired production of hamartin or tuberin proteins, leading to the manifestation of skin lesions, tumors, and seizures. TSC can manifest in multiple organ systems with the cutaneous and renal systems being the most commonly affected. These manifestations can secondarily lead to the development of hypertension, chronic kidney disease, and neurocognitive declines. The renal pathologies most commonly seen in TSC are angiomyolipoma, renal cysts, and less commonly, oncocytomas. In this review, we highlight the current understanding on the renal manifestations of TSC along with current diagnosis and treatment guidelines.
    Keywords:  Von Hippel–Lindau disease; angiomyolipoma; autosomal polycystic kidney disease; renal cystic disease; tuberous sclerosis
    DOI:  https://doi.org/10.15586/jkcvhl.2020.131
  41. EMBO Rep. 2020 Sep 23. e50635
    Altea-Manzano P, Cuadros AM, Broadfield LA, Fendt SM.
      Nutrients are indispensable resources that provide the macromolecular building blocks and energy requirements for sustaining cell growth and survival. Cancer cells require several key nutrients to fulfill their changing metabolic needs as they progress through stages of development. Moreover, both cell-intrinsic and microenvironment-influenced factors determine nutrient dependencies throughout cancer progression-for which a comprehensive characterization remains incomplete. In addition to the widely studied role of genetic alterations driving cancer metabolism, nutrient use in cancer tissue may be affected by several factors including the following: (i) diet, the primary source of bodily nutrients which influences circulating metabolite levels; (ii) tissue of origin, which can influence the tumor's reliance on specific nutrients to support cell metabolism and growth; (iii) local microenvironment, which dictates the accessibility of nutrients to tumor cells; (iv) tumor heterogeneity, which promotes metabolic plasticity and adaptation to nutrient demands; and (v) functional demand, which intensifies metabolic reprogramming to fuel the phenotypic changes required for invasion, growth, or survival. Here, we discuss the influence of these factors on nutrient metabolism and dependence during various steps of tumor development and progression.
    Keywords:  cancer metabolism; diet; microenvironment; nutrients; tumor heterogeneity
    DOI:  https://doi.org/10.15252/embr.202050635
  42. Front Neurosci. 2020 ;14 899
    Gelot AB, Represa A.
      Tuberous sclerosis (TSC) is a multisystem autosomal dominant genetic disorder due to loss of function of TSC1/TSC2 resulting in increased mTOR (mammalian target of rapamycin) signaling. In the brain, TSC is characterized by the formation of specific lesions that include subependymal and white matter nodules and cortical tubers. Cells that constitute TSC lesions are mainly Giant cells and dysmorphic neurons and astrocytes, but normal cells also populate the tubers. Although considered as a developmental disorder, the histopathological features of brain lesions have been described in only a limited number of fetal cases, providing little information on how these lesions develop. In this report we characterized the development of TSC lesions in 14 fetal brains ranging from 19 gestational weeks (GW) to term and 2 postnatal cases. The study focused on the telencephalon at the level of the caudothalamic notch. Our data indicate that subcortical lesions, forming within and at the vicinity of germinative zones, are the first alterations (already detected in 19GW brains), characterized by the presence of numerous dysmorphic astrocytes and Giant, balloon-like, cells. Our data show that cortical tuber formation is a long process that initiates with the presence of dysmorphic astrocytes (by 19-21GW), progress with the apparition of Giant cells (by 24GW) and mature with the appearance of dysmorphic neurons by the end of gestation (by 36GW). Furthermore, the typical tuberal aspect of cortical lesions is only reached when bundles of neurofilament positive extensions delineate the bottom of the cortical lesion (by 36GW). In addition, our study reveals the presence of Giant cells and dysmorphic neurons immunopositive for interneuron markers such as calbindin and parvalbumin, suggesting that TSC lesions would be mosaic lesions generated from different classes of progenitors.
    Keywords:  Giant cells; development; dysmorphic neurons; interneurons; tubers
    DOI:  https://doi.org/10.3389/fnins.2020.00899