bims-proteo Biomed News
on Proteostasis
Issue of 2020‒05‒10
twenty-four papers selected by
Eric Chevet
INSERM


  1. Liver Int. 2020 May 06.
    Santos-Laso A, Izquierdo-Sanchez L, Rodrigues PM, Huang BQ, Azkargorta M, Lapitz A, Munoz-Garrido P, Arbelaiz A, Caballero-Camino FJ, Fernández-Barrena MG, Jimenez-Agüero R, Argemi J, Aragon T, Elortza F, Marzioni M, Drenth JPH, LaRusso NF, Bujanda L, Perugorria MJ, Banales JM.
      BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target.METHODS: ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro.
    RESULTS: The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis.
    CONCLUSIONS: Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.
    Keywords:  cholangiocyte; endoplasmic reticulum stress; hepatic cystogenesis; pathogenesis; proteostasis; unfolded protein response
    DOI:  https://doi.org/10.1111/liv.14485
  2. J Biol Chem. 2020 May 06. pii: jbc.RA119.012280. [Epub ahead of print]
    Alam SMD, Tsukamoto Y, Ogawa M, Senoo Y, Ikeda K, Tashima Y, Takeuchi H, Okajima T.
      Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analyses revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its ER localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with wildtype EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.
    Keywords:  EOGT; N-linked glycosylation; Notch receptor; O-linked N-acetylglucosamine (O-GlcNAc); endoplasmic reticulum (ER); glycoprotein; oligomannose glycan; posttranslational modification; protein folding; protein maturation
    DOI:  https://doi.org/10.1074/jbc.RA119.012280
  3. J Biol Chem. 2020 May 06. pii: jbc.RA120.012835. [Epub ahead of print]
    Tang X, Zhang L, Ma T, Wang M, Li B, Jiang L, Yan Y, Guo Y.
      Planar cell polarity (PCP) is a process during which cells are polarized along the plane of the epithelium and is regulated by several transmembrane signaling proteins. After their synthesis, these PCP proteins are delivered along the secretory transport pathway to the plasma membrane, where they perform their physiological functions. However, the molecular mechanisms that regulate PCP protein transport remain largely unclear. We found here that the delivery of a PCP protein, Frizzled-6, to the cell surface is regulated by two conserved polybasic motifs: one located in its first intracellular loop and the other in its C-terminal cytosolic domain. We observed that the polybasic motif of Frizzled is also important for its surface localization in the Drosophila wing. Results from a mechanistic analysis indicated that Frizzled-6 packaging into vesicles at the endoplasmic reticulum (ER) is regulated by a direct interaction between the polybasic motif and the Glu-62 and Glu-63 residues on the secretion-associated Ras-related GTPase 1A (SAR1A) subunit of coat protein complex II (COPII). Moreover, we found that newly synthesized Frizzled-6 is associated with another PCP protein, cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), in the secretory transport pathway and that this association regulates their surface delivery. Our results reveal insight into the molecular machinery that regulates the ER export of Frizzled-6. They also suggest that the association of CELSR1 with Frizzled-6 is important, enabling efficient Frizzled-6 delivery to the cell surface, providing a quality-control mechanism that ensures appropriate stoichiometry of these two PCP proteins at cell boundaries.
    Keywords:  Golgi; endoplasmic reticulum (ER); protein sorting; trafficking; vesicles
    DOI:  https://doi.org/10.1074/jbc.RA120.012835
  4. J Biol Chem. 2020 May 05. pii: jbc.RA119.011222. [Epub ahead of print]
    Nagata A, Itoh F, Sasho A, Sugita K, Suzuki R, Hinata H, Shimoda Y, Suzuki E, Maemoto Y, Inagawa T, Fujikawa Y, Ikeda E, Fujii C, Inoue H.
      Modification of the transforming growth factor β (TGF-β) signaling components by (de)ubiquitination is emerging as a key regulatory mechanism that controls cell signaling responses in health and disease. Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7/TGF-β signaling, suggesting that this mode of regulation of TGF-β signaling is conserved across animal species. The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7/TGF-β signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1/TGF-βRI, and daf4/R-SMAD, but not of daf-8/R-SMAD. This suggested that UBH-1 may stimulate DAF-7/TGF-β signaling via DAF-8/R-SMAD. Therefore, we investigated the effect of UCH-L1 on TGF-β signaling via its intracellular effectors, i.e. SMAD2 and SMAD3, in mammalian cells. Overexpression of UCH-L1, but not of UCH-L3 (the other human homolog of UBH1) or of the catalytic mutant UCH-L1C90A, enhanced TGF-β/SMAD-induced transcriptional activity, indicating that the deubiquitination activity of UCH-L1 is indispensable for enhancing TGF-β/SMAD signaling. We also found that UCH-L1 interacts, deubiquitinates, and stabilizes SMAD2 and SMAD3. Under hypoxia, UCH-L1 expression increased and TGF-β/SMAD signaling was potentiated in the A549 human lung adenocarcinoma cell line. Notably, UCH-L1-deficient A549 cells were impaired in tumorigenesis, and, unlike wildtype UCH-L1, a UCH-L1 variant lacking deubiquitinating activity was unable to restore tumorigenesis in these cells. These results indicate that UCH-L1 activity supports DAF-7/TGF-β signaling and suggest that UCH-L1's deubiquitination activity is a potential therapeutic target for managing lung cancer.
    Keywords:  DAF-7; SMAD transcription factor; deubiquitylation (deubiquitination); hypoxia; lung cancer; transforming growth factor beta (TGF-B); ubh-1/UCH-L1
    DOI:  https://doi.org/10.1074/jbc.RA119.011222
  5. EMBO Rep. 2020 May 03. e48927
    Govindarajan S, Verheugen E, Venken K, Gaublomme D, Maelegheer M, Cloots E, Gysens F, De Geest BG, Cheng TY, Moody DB, Janssens S, Drennan M, Elewaut D.
      CD1d-restricted invariant natural killer T (iNKT) cells constitute a common glycolipid-reactive innate-like T-cell subset with a broad impact on innate and adaptive immunity. While several microbial glycolipids are known to activate iNKT cells, the cellular mechanisms leading to endogenous CD1d-dependent glycolipid responses remain largely unclear. Here, we show that endoplasmic reticulum (ER) stress in APCs is a potent inducer of CD1d-dependent iNKT cell autoreactivity. This pathway relies on the presence of two transducers of the unfolded protein response: inositol-requiring enzyme-1a (IRE1α) and protein kinase R-like ER kinase (PERK). Surprisingly, the neutral but not the polar lipids generated within APCs undergoing ER stress are capable of activating iNKT cells. These data reveal that ER stress is an important mechanism to elicit endogenous CD1d-restricted iNKT cell responses through induction of distinct classes of neutral lipids.
    Keywords:  ER stress; NKT cells; antigen-presenting cells; lipid antigens; neutral lipid
    DOI:  https://doi.org/10.15252/embr.201948927
  6. Proc Natl Acad Sci U S A. 2020 May 04. pii: 202003043. [Epub ahead of print]
    Oh JH, Hyun JY, Chen SJ, Varshavsky A.
      The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal (Nt) residues. If a substrate bears, for example, Nt-Asn, its targeting involves deamidation of Nt-Asn, arginylation of resulting Nt-Asp, binding of resulting (conjugated) Nt-Arg to the UBR1-RAD6 E3-E2 ubiquitin ligase, ligase-mediated synthesis of a substrate-linked polyubiquitin chain, its capture by the proteasome, and substrate's degradation. We discovered that the human Nt-Asn-specific Nt-amidase NTAN1, Nt-Gln-specific Nt-amidase NTAQ1, arginyltransferase ATE1, and the ubiquitin ligase UBR1-UBE2A/B (or UBR2-UBE2A/B) form a complex in which NTAN1 Nt-amidase binds to NTAQ1, ATE1, and UBR1/UBR2. In addition, NTAQ1 Nt-amidase and ATE1 arginyltransferase also bind to UBR1/UBR2. In the yeast Saccharomyces cerevisiae, the Nt-amidase, arginyltransferase, and the double-E3 ubiquitin ligase UBR1-RAD6/UFD4-UBC4/5 are shown to form an analogous targeting complex. These complexes may enable substrate channeling, in which a substrate bearing, for example, Nt-Asn, would be captured by a complex-bound Nt-amidase, followed by sequential Nt modifications of the substrate and its polyubiquitylation at an internal Lys residue without substrate's dissociation into the bulk solution. At least in yeast, the UBR1/UFD4 ubiquitin ligase interacts with the 26S proteasome, suggesting an even larger Arg/N-degron-targeting complex that contains the proteasome as well. In addition, specific features of protein-sized Arg/N-degron substrates, including their partly sequential and partly nonsequential enzymatic modifications, led us to a verifiable concept termed "superchanneling." In superchanneling, the synthesis of a substrate-linked poly-Ub chain can occur not only after a substrate's sequential Nt modifications, but also before them, through a skipping of either some or all of these modifications within a targeting complex.
    Keywords:  channeling; degradation; degron; superchanneling; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.2003043117
  7. Lab Invest. 2020 May 04.
    Mahadevan J, Morikawa S, Yagi T, Abreu D, Lu S, Kanekura K, Brown CM, Urano F.
      Endoplasmic reticulum (ER) stress-mediated cell death is an emerging target for human chronic disorders, including neurodegeneration and diabetes. However, there is currently no treatment for preventing ER stress-mediated cell death. Here, we show that mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor secreted from ER stressed cells, prevents ER stress-mediated β cell death and enhances β cell proliferation in cell and mouse models of Wolfram syndrome, a prototype of ER disorders. Our results indicate that molecular pathways regulated by MANF are promising therapeutic targets for regenerative therapy of ER stress-related disorders, including diabetes, retinal degeneration, neurodegeneration, and Wolfram syndrome.
    DOI:  https://doi.org/10.1038/s41374-020-0436-1
  8. Cell. 2020 Apr 29. pii: S0092-8674(20)30404-9. [Epub ahead of print]
    Iserman C, Desroches Altamirano C, Jegers C, Friedrich U, Zarin T, Fritsch AW, Mittasch M, Domingues A, Hersemann L, Jahnel M, Richter D, Guenther UP, Hentze MW, Moses AM, Hyman AA, Kramer G, Kreysing M, Franzmann TM, Alberti S.
      Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.
    Keywords:  5′; Ded1p; UTR; chaperone; condensate; cytosolic pH; evolutionary adaptation; heat shock response; heat stress; phase separation; ribosomal scanning
    DOI:  https://doi.org/10.1016/j.cell.2020.04.009
  9. Mol Psychiatry. 2020 May 04.
    Xu Y, Du S, Marsh JA, Horie K, Sato C, Ballabio A, Karch CM, Holtzman DM, Zheng H.
      Neurofibrillary tangles (NFTs) composed of hyperphosphorylated and misfolded tau protein are a pathological hallmark of Alzheimer's disease and other tauopathy conditions. Tau is predominantly an intraneuronal protein but is also secreted in physiological and pathological conditions. The extracellular tau has been implicated in the seeding and propagation of tau pathology and is the prime target of the current tau immunotherapy. However, truncated tau species lacking the microtubule-binding repeat (MTBR) domains essential for seeding have been shown to undergo active secretion and the mechanisms and functional consequences of the various extracellular tau are poorly understood. We report here that the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, plays an essential role in the lysosomal exocytosis of selected tau species. TFEB loss of function significantly reduced the levels of interstitial fluid (ISF) tau in PS19 mice expressing P301S mutant tau and in conditioned media of mutant tau expressing primary neurons, while the secretion of endogenous wild-type tau was not affected. Mechanistically we found that TFEB regulates the secretion of truncated mutant tau lacking MTBR and this process is dependent on the lysosomal calcium channel TRPML1. Consistent with the seeding-incompetent nature of the truncated tau and supporting the concept that TFEB-mediated lysosomal exocytosis promotes cellular clearance, we show that reduced ISF tau in the absence of TFEB is associated with enhanced intraneuronal pathology and accelerated spreading. Our results support the idea that TFEB-mediated tau exocytosis serves as a clearance mechanism to reduce intracellular tau under pathological conditions and that effective tau immunotherapy should devoid targeting these extracellular tau species.
    DOI:  https://doi.org/10.1038/s41380-020-0738-0
  10. J Cell Sci. 2020 May 06. pii: jcs.239822. [Epub ahead of print]
    O'Loughlin T, Kruppa AJ, Ribeiro ALR, Edgar JR, Ghannam A, Smith AM, Buss F.
      Optineurin (OPTN) is a multifunctional protein involved in autophagy, secretion as well as NF-κB and IRF3 signalling and OPTN mutations are associated with several human diseases. Here we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-κB and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for ATG9A. Disease mutations linked to POAG cause aberrant foci formation in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. Using proximity labelling proteomics, we identify the LUBAC complex, CYLD and TBK1 as part of the OPTN interactome and show that these proteins are recruited to this OPTN-positive perinuclear compartment. Our work uncovers a crucial role for OPTN in dampening NF-κB and IRF3 signalling through the sequestration of LUBAC and other positive regulators in this viral RNA-induced compartment leading to altered pro-inflammatory cytokine secretion.
    Keywords:  ATG9A; BioID; Functional proteomics; IFN; LUBAC; Linear ubiquitin; NF-κB; OPTN; TBK1
    DOI:  https://doi.org/10.1242/jcs.239822
  11. Cells. 2020 May 04. pii: E1131. [Epub ahead of print]9(5):
    Trivedi PC, Bartlett JJ, Pulinilkunnil T.
      Lysosomes are the main proteolytic compartments of mammalian cells comprising of a battery of hydrolases. Lysosomes dispose and recycle extracellular or intracellular macromolecules by fusing with endosomes or autophagosomes through specific waste clearance processes such as chaperone-mediated autophagy or microautophagy. The proteolytic end product is transported out of lysosomes via transporters or vesicular membrane trafficking. Recent studies have demonstrated lysosomes as a signaling node which sense, adapt and respond to changes in substrate metabolism to maintain cellular function. Lysosomal dysfunction not only influence pathways mediating membrane trafficking that culminate in the lysosome but also govern metabolic and signaling processes regulating protein sorting and targeting. In this review, we describe the current knowledge of lysosome in influencing sorting and nutrient signaling. We further present a mechanistic overview of intra-lysosomal processes, along with extra-lysosomal processes, governing lysosomal fusion and fission, exocytosis, positioning and membrane contact site formation. This review compiles existing knowledge in the field of lysosomal biology by describing various lysosomal events necessary to maintain cellular homeostasis facilitating development of therapies maintaining lysosomal function.
    Keywords:  autophagy; calcium; cathepsin; endocytosis; lysosome; mannose-6-phosphate; metabolism; proton
    DOI:  https://doi.org/10.3390/cells9051131
  12. Cancers (Basel). 2020 Apr 29. pii: E1113. [Epub ahead of print]12(5):
    Lafont E.
      Throughout tumour progression, tumour cells are exposed to various intense cellular stress conditions owing to intrinsic and extrinsic cues, to which some cells are remarkably able to adapt. Death Receptor (DR) signalling and the Unfolded Protein Response (UPR) are two stress responses that both regulate a plethora of outcomes, ranging from proliferation, differentiation, migration, cytokine production to the induction of cell death. Both signallings are major modulators of physiological tissue homeostasis and their dysregulation is involved in tumorigenesis and the metastastic process. The molecular determinants of the control between the different cellular outcomes induced by DR signalling and the UPR in tumour cells and their stroma and their consequences on tumorigenesis are starting to be unravelled. Herein, I summarize the main steps of DR signalling in relation to its cellular and pathophysiological roles in cancer. I then highlight how the UPR and DR signalling control common cellular outcomes and also cross-talk, providing potential opportunities to further understand the development of malignancies.
    Keywords:  CD95; ER stress; IRE1; PERK; TNFR1; TRAIL-R1/2; cell death; death receptor; post-translational modifications; unfolded protein response
    DOI:  https://doi.org/10.3390/cancers12051113
  13. Front Cell Infect Microbiol. 2020 ;10 149
    Malvezzi AM, Aricó M, Souza-Melo N, Dos Santos GP, Bittencourt-Cunha P, Holetz FB, Schenkman S.
      The integrated stress response in eukaryotic cells is an orchestrated pathway that leads to eukaryotic Initiation Factor 2 alpha subunit (eIF2α) phosphorylation at ser51 and ultimately activates pathways to mitigate cellular damages. Three putative kinases (Tck1, Tck2, and Tck3) are found in the Trypanosoma cruzi genome, the flagellated parasite that causes Chagas disease. These kinases present similarities to other eukaryotic eIF2α kinases, exhibiting a typical insertion loop in the kinase domain of the protein. We found that this insertion loop is conserved among kinase 1 of several T. cruzi strains but differs among various Kinetoplastidae species, suggesting unique roles. Kinase 1 is orthologous of GCN2 of several eukaryotes, which have been implicated in the eIF2α ser51 phosphorylation in situations that mainly affects the nutrients levels. Therefore, we further investigated the responses to nutritional stress of T. cruzi devoid of TcK1 generated by CRISPR/Cas9 gene replacement. In nutrient-rich conditions, replicative T. cruzi epimastigotes depleted of TcK1 proliferate as wild type cells but showed increased levels of polysomes relative to monosomes. Upon nutritional deprivation, the polysomes decreased more than in TcK1 depleted line. However, eIF2α is still phosphorylated in TcK1 depleted line, as in wild type parasites. eIF2α phosphorylation increased at longer incubations times, but KO parasites showed less accumulation of ribonucleoprotein granules containing ATP-dependent RNA helicase involved in mRNA turnover (DHH1) and Poly-A binding protein (PABP1). Additionally, the formation of metacyclic-trypomastigotes is increased in the absence of Tck1 compared to controls. These metacyclics, as well as tissue culture trypomastigotes derived from the TcK1 knockout line, were less infective to mammalian host cells, although replicated faster inside mammalian cells. These results indicate that GCN2-like kinase in T. cruzi affects stress granule formation, independently of eIF2α phosphorylation upon nutrient deprivation. It also modulates the fate of the parasites during differentiation, invasion, and intracellular proliferation.
    Keywords:  CRISPR/Cas9; GCN2; TcK1; Trypanosoma cruzi; eIF2α; phosphorylation; stress-granules
    DOI:  https://doi.org/10.3389/fcimb.2020.00149
  14. Histochem Cell Biol. 2020 May 07.
    Daverkausen-Fischer L, Motyl-Eisemann M, Draga M, Scaal M, Pröls F.
      The vertebrate-specific co-chaperone Mdg1/ERdj4, which is localized in the endoplasmic reticulum, controls the folding and degradation of proteins. We characterized its protein pattern during chick embryonic development. During early development, Mdg1/ERdj4 protein is present in mesenchymal and epithelial cells. In mesenchymal cells, it has a salt and pepper pattern. In contrast, during epithelial tissue differentiation, Mdg1/ERdj4 marks the basal and/or apical compartment of epithelial linings. The distinct protein pattern in epithelial tissue might point to its role in organizing and maintaining the epithelial structure. This could be achieved, e.g. by controlling folding and secretion of membrane-bound receptors or by inhibiting the IRE1α-Xbp1s-SNAI1/2-induced mesenchymalization. High Mdg1/ERdj4 protein levels are maintained in tissue with sustained secretory activity as in ependymal cells or enterocytes, substantiating its important role for secretion. We conclude that the transient elevation of Mdg1/ERdj4 protein levels controls the differentiation of epithelial linings while constitutive high levels are closely linked to secretory activity.
    Keywords:  Chick; Epithelial–mesenchymal transition (EMT); Hsp40 protein; Mdg1/ERdj4/mDjB9; Mesenchymal–epithelial transition (MET); Molecular chaperone
    DOI:  https://doi.org/10.1007/s00418-020-01881-x
  15. EMBO Rep. 2020 May 08. e49117
    Ciscato F, Filadi R, Masgras I, Pizzi M, Marin O, Damiano N, Pizzo P, Gori A, Frezzato F, Chiara F, Trentin L, Bernardi P, Rasola A.
      Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.
    Keywords:  Hexokinase 2; anti-neoplastic strategy; cancer; cell penetrating peptide; mitochondria-associated membranes
    DOI:  https://doi.org/10.15252/embr.201949117
  16. Blood. 2020 May 08. pii: blood.2019004276. [Epub ahead of print]
    Lo R, Li L, Pluthero FG, Leung R, Eto K, Kahr WH.
      Studies of inherited platelet disorders have provided many insights into platelet development and function. Loss of function of neurobeachin-like 2 (NBEAL2) causes Gray Platelet Syndrome (GPS), where the absence of platelet α-granules indicates NBEAL2 is required for their production by precursor megakaryocytes. The endoplasmic reticulum is a dynamic network that interacts with numerous intracellular vesicles and organelles and plays key roles in their development. The megakaryocyte endoplasmic reticulum is very extensive, and in this study we investigated its role in the biogenesis of α-granules by focusing on the membrane-resident trafficking protein SEC22B. Co-immunoprecipitation experiments using tagged proteins expressed in human HEK293 and megakaryocytic imMKCL cells established binding of NBEAL2 with SEC22B, and demonstrated that NBEAL2 can simultaneously bind SEC22B and P-selectin. NBEAL2-SEC22B binding was also observed for endogenous proteins in human megakaryocytes using co-immunoprecipitation, and immunofluorescence microscopy detected substantial overlap. SEC22B binding was localized to a region of NBEAL2 spanning amino acids 1798-1903, where two GPS-associated missense variants have been reported: E1833K and R1839C. NBEAL2 containing either variant did not bind SEC22B co-expressed in HEK292 cells. CRISPR/Cas9 mediated knockout of SEC22B in imMKCL cells resulted in decreased NBEAL2, but not vice versa. Loss of either SEC22B or NBEAL2 expression also resulted in failure of α-granule production and reduced granule proteins in imMKCL cells. We conclude that SEC22B is required for α-granule biogenesis in megakaryocytes, and that interactions with SEC22B and P-selectin facilitate the essential role of NBEAL2 in granule development and cargo stability.
    DOI:  https://doi.org/10.1182/blood.2019004276
  17. Mol Cell. 2020 Apr 24. pii: S1097-2765(20)30236-7. [Epub ahead of print]
    Levine DC, Hong H, Weidemann BJ, Ramsey KM, Affinati AH, Schmidt MS, Cedernaes J, Omura C, Braun R, Lee C, Brenner C, Peek CB, Bass J.
      Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here, we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and that is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.
    Keywords:  NAD(+); SIRT1; aging; circadian; clock; heat shock factor 1; liver; nicotinamide mononucleotide; nicotinamide riboside; transcriptomics
    DOI:  https://doi.org/10.1016/j.molcel.2020.04.010
  18. Cell Rep. 2020 May 05. pii: S2211-1247(20)30558-1. [Epub ahead of print]31(5): 107609
    Lockridge A, Jo S, Gustafson E, Damberg N, Mohan R, Olson M, Abrahante JE, Alejandro EU.
      During early obesity, pancreatic β cells compensate for increased metabolic demand through a transient phase of insulin hypersecretion that stabilizes blood glucose and forestalls diabetic progression. We find evidence that β cell O-GlcNAcylation, a nutrient-responsive post-translational protein modification regulated by O-GlcNAc transferase (OGT), is critical for coupling hyperlipidemia to β cell functional adaptation during this compensatory prediabetic phase. In mice, islet O-GlcNAcylation rises and falls in tandem with the timeline of secretory potentiation during high-fat feeding while genetic models of β-cell-specific OGT loss abolish hyperinsulinemic responses to lipids, in vivo and in vitro. We identify the endoplasmic reticulum (ER) Ca2+ ATPase SERCA2 as a β cell O-GlcNAcylated protein in mice and humans that is able to rescue palmitate-stimulated insulin secretion through pharmacological activation. This study reveals an important physiological role for β cell O-GlcNAcylation in sensing and responding to obesity, with therapeutic implications for managing the relationship between type 2 diabetes and its most common risk factor.
    DOI:  https://doi.org/10.1016/j.celrep.2020.107609
  19. J Invest Dermatol. 2020 Apr 29. pii: S0022-202X(20)31404-4. [Epub ahead of print]
    Avitan-Hersh E, Feng Y, Vaisman AO, Ahmed YA, Zohar Y, Zhang T, Lee JS, Lazar I, Khalil SS, Feiler Y, Kluger H, Kahana C, Brown K, Ruppin E, Ronai ZA, Orian A.
      Among the hallmarks of melanoma, are impaired proteostasis and the rapid development of resistance to targeted therapy, that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties including therapy resistance require the translation initiation factor eIF2α. RNF4 binds, ubiquitinates and stabilizes the phosphorylated eIF2α (p-eIF2α), but not ATF4 or CHOP that mediate eIF2α-dependent integrated stress response. In accordance, p-eIF2α level was significantly elevated in high-RNF4 patient derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.
    Keywords:  Cancer; Gene-regulation; Melanoma; RNF4; STUbL; Translation; Ubiquitin; p-eIF2α
    DOI:  https://doi.org/10.1016/j.jid.2020.04.008
  20. Cell Rep. 2020 May 05. pii: S2211-1247(20)30559-3. [Epub ahead of print]31(5): 107610
    Han P, Shichino Y, Schneider-Poetsch T, Mito M, Hashimoto S, Udagawa T, Kohno K, Yoshida M, Mishima Y, Inada T, Iwasaki S.
      Ribosome movement is not always smooth and is rather often impeded. For ribosome pauses, fundamental issues remain to be addressed, including where ribosomes pause on mRNAs, what kind of RNA/amino acid sequence causes this pause, and the physiological significance of this attenuation of protein synthesis. Here, we survey the positions of ribosome collisions caused by ribosome pauses in humans and zebrafish using modified ribosome profiling. Collided ribosomes, i.e., disomes, emerge at various sites: Pro-Pro/Gly/Asp motifs; Arg-X-Lys motifs; stop codons; and 3' untranslated regions. The electrostatic interaction between the charged nascent chain and the ribosome exit tunnel determines the eIF5A-mediated disome rescue at the Pro-Pro sites. In particular, XBP1u, a precursor of endoplasmic reticulum (ER)-stress-responsive transcription factor, shows striking queues of collided ribosomes and thus acts as a degradation substrate by ribosome-associated quality control. Our results provide insight into the causes and consequences of ribosome pause by dissecting collided ribosomes.
    Keywords:  XBP; disome; disome profiling; eIF5A; ribosome; ribosome collision; ribosome profiling; ribosome-associated quality control (RQC); translation; translation elongation
    DOI:  https://doi.org/10.1016/j.celrep.2020.107610
  21. FEBS Lett. 2020 May 02.
    Ramesh R, Sattlegger E.
      The signalling pathway governing Gcn2 kinase allows cells to cope with amino acid shortage. Under starvation, Gcn2 phosphorylates the translation initiation factor eIF2α, triggering downstream events that ultimately allow cells to cope with starvation. Under nutrient-replete conditions, the translation elongation factor eEF1A binds Gcn2 to contribute to keeping Gcn2 inactive. Here, we aimed to map the regions in eEF1A involved in binding and/or regulating Gcn2. We find that eEF1A amino acids 1-221 and 222-315, containing most of domains I and II, respectively, bind Gcn2 in vitro. Overexpression of eEF1A lacking or containing domain III, impairs eIF2α phosphorylation. While the latter reduces growth under starvation similarly to eEF1A lacking domain I, the former enhances growth in a Gcn2-dependent manner. Our studies suggest that domain II is required for Gcn2-inhibition and that eEF1A lacking domain III mainly affects the Gcn2-response pathway downstream of Gcn2.
    Keywords:  Gcn2; General control non-derepressible; amino acid starvation; eEF1A; eukaryotic translation elongation factor; protein-protein interaction
    DOI:  https://doi.org/10.1002/1873-3468.13803
  22. Science. 2020 May 04. pii: eabb9983. [Epub ahead of print]
    Watanabe Y, Allen JD, Wrapp D, McLellan JS, Crispin M.
      The emergence of the betacoronavirus, SARS-CoV-2, the causative agent of COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.
    DOI:  https://doi.org/10.1126/science.abb9983
  23. Elife. 2020 May 05. pii: e52979. [Epub ahead of print]9
    Chen Y, Kotian N, Aranjuez G, Chen L, Messer CL, Burtscher A, Sawant K, Ramel D, Wang X, McDonald JA.
      Collective cell migration is central to many developmental and pathological processes. However, the mechanisms that keep cell collectives together and coordinate movement of multiple cells are poorly understood. Using the Drosophila border cell migration model, we find that Protein phosphatase 1 (Pp1) activity controls collective cell cohesion and migration. Inhibition of Pp1 causes border cells to round up, dissociate, and move as single cells with altered motility. We present evidence that Pp1 promotes proper levels of cadherin-catenin complex proteins at cell-cell junctions within the cluster to keep border cells together. Pp1 further restricts actomyosin contractility to the cluster periphery rather than at individual internal border cell contacts. We show that the myosin phosphatase Pp1 complex, which inhibits non-muscle myosin-II (Myo-II) activity, coordinates border cell shape and cluster cohesion. Given the high conservation of Pp1 complexes, this study identifies Pp1 as a major regulator of collective versus single cell migration.
    Keywords:  D. melanogaster; cell adhesion; cell biology; cell collective; cell migration; developmental biology; myosin; protein phosphatases
    DOI:  https://doi.org/10.7554/eLife.52979
  24. Sci Transl Med. 2020 May 06. pii: eaba0769. [Epub ahead of print]12(542):
    Ju D, Zhang W, Yan J, Zhao H, Li W, Wang J, Liao M, Xu Z, Wang Z, Zhou G, Mei L, Hou N, Ying S, Cai T, Chen S, Xie X, Lai L, Tang C, Park N, Takahashi JS, Huang N, Qi X, Zhang EE.
      Transcriptional regulation lies at the core of the circadian clockwork, but how the clock-related transcription machinery controls the circadian phase is not understood. Here, we show both in human cells and in mice that RuvB-like ATPase 2 (RUVBL2) interacts with other known clock proteins on chromatin to regulate the circadian phase. Pharmacological perturbation of RUVBL2 with the adenosine analog compound cordycepin resulted in a rapid-onset 12-hour clock phase-shift phenotype at human cell, mouse tissue, and whole-animal live imaging levels. Using simple peripheral injection treatment, we found that cordycepin penetrated the blood-brain barrier and caused rapid entrainment of the circadian phase, facilitating reduced duration of recovery in a mouse jet-lag model. We solved a crystal structure for human RUVBL2 in complex with a physiological metabolite of cordycepin, and biochemical assays showed that cordycepin treatment caused disassembly of an interaction between RUVBL2 and the core clock component BMAL1. Moreover, we showed with spike-in ChIP-seq analysis and binding assays that cordycepin treatment caused disassembly of the circadian super-complex, which normally resides at E-box chromatin loci such as PER1, PER2, DBP, and NR1D1 Mathematical modeling supported that the observed type 0 phase shifts resulted from derepression of E-box clock gene transcription.
    DOI:  https://doi.org/10.1126/scitranslmed.aba0769