bims-proteo Biomed News
on Proteostasis
Issue of 2020‒11‒01
fifty-nine papers selected by
Eric Chevet
INSERM


  1. Autophagy. 2020 Oct 28. 1-17
    Zielke S, Kardo S, Zein L, Mari M, Covarrubias-Pinto A, Kinzler MN, Meyer N, Stolz A, Fulda S, Reggiori F, Kögel D, van Wijk SJL.
      Selective degradation of the endoplasmic reticulum (ER; reticulophagy) is a type of autophagy involved in the removal of ER fragments. So far, amino acid starvation as well as ER stress have been described as inducers of reticulophagy, which in turn restores cellular energy levels and ER homeostasis. Here, we explored the autophagy-inducing mechanisms that underlie the autophagic cell death (ACD)-triggering compound loperamide (LOP) in glioblastoma cells. Interestingly, LOP triggers upregulation of the transcription factor ATF4, which is accompanied by the induction of additional ER stress markers. Notably, knockout of ATF4 significantly attenuated LOP-induced autophagy and ACD. Functionally, LOP also specifically induces the engulfment of large ER fragments within autophagosomes and lysosomes as determined by electron and fluorescence microscopy. LOP-induced reticulophagy and cell death are predominantly mediated through the reticulophagy receptor RETREG1/FAM134B and, to a lesser extent, TEX264, confirming that reticulophagy receptors can promote ACD. Strikingly, apart from triggering LOP-induced autophagy and ACD, ATF4 is also required for LOP-induced reticulophagy. These observations highlight a key role for ATF4, RETREG1 and TEX264 in response to LOP-induced ER stress, reticulophagy and ACD, and establish a novel mechanistic link between ER stress and reticulophagy, with possible implications for additional models of drug-induced ER stress. Abbreviations: ACD: autophagic cell death; ATF6: activating transcription factor 6; ATL3: atlastin 3; BafA1: bafilomycin A1; CCPG1: cell cycle progression gene 1; co-IP: co-immunoprecipitation; DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; GBM: glioblastoma multiforme; HSPA5/BiP: heat shock protein family (Hsp70) member 5; LOP: loperamide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; RETREG1/FAM134B: reticulophagy regulator 1; RTN3L: reticulon 3 long; SEC62: SEC62 homolog, protein translocation factor; TEX264: testis-expressed 264, reticulophagy receptor; UPR: unfolded protein response.
    Keywords:  HSPA5/BiP; MEFs; MZ-54; RETREG1/FAM134B; TEX264; autophagic cell death; loperamide; p-eIF2α; selective autophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1827780
  2. Aging Cell. 2020 Oct 31. e13265
    Taylor RC, Hetz C.
      The aging process is characterized by a progressive decline in the function of most tissues, representing the main risk factor in the development of a variety of human diseases. Studies in multiple animal models have demonstrated that interventions that improve the capacity to maintain endoplasmic reticulum (ER) proteostasis prolong life and healthspan. ER stress is monitored by the unfolded protein response (UPR), a signaling pathway that mediates adaptive processes to restore proteostasis or the elimination of damaged cells by apoptosis. Here, we discuss recent advances in understanding the significance of the UPR to aging and its implications for the maintenance of cell physiology of various cell types and organs. The possible benefits of targeting the UPR to extend healthspan and reduce the risk of developing age-related diseases are also discussed.
    Keywords:  ER stress; aging; autophagy; cell-nonautonomous; protein misfolding; proteostasis
    DOI:  https://doi.org/10.1111/acel.13265
  3. Autophagy. 2020 Oct 28. 1-16
    Chou HY, Lee YT, Lin YJ, Wen JK, Peng WH, Hsieh PL, Lin SY, Hung CC, Chen GC.
      Macroautophagy/autophagy is an evolutionarily conserved intracellular pathway for the degradation of cytoplasmic materials. Under stress conditions, autophagy is upregulated and double-membrane autophagosomes are formed by the expansion of phagophores. The ATG16L1 precursor fusion contributes to development of phagophore structures and is critical for the biogenesis of autophagosomes. Here, we discovered a novel role of the protein tyrosine phosphatase PTPN9 in the regulation of homotypic ATG16L1 vesicle fusion and early autophagosome formation. Depletion of PTPN9 and its Drosophila homolog Ptpmeg2 impaired autophagosome formation and autophagic flux. PTPN9 colocalized with ATG16L1 and was essential for homotypic fusion of ATG16L1+ vesicles during starvation-induced autophagy. We further identified the Q-SNARE VTI1B as a substrate target of PTPN9 phosphatase. Like PTPN9, the VTI1B nonphosphorylatable mutant but not the phosphomimetic mutant enhanced SNARE complex assembly and autophagic flux. Our findings highlight the important role of PTPN9 in the regulation of ATG16L1+ autophagosome precursor fusion and autophagosome biogenesis through modulation of VTI1B phosphorylation status. Abbreviations: csw: corkscrew; EBSS: Earle's balanced salt solution; ERGIC: ER-Golgi intermediate compartment; ESCRT: endosomal sorting complexes required for transport; mop: myopic; NSF: N-ethylmaleimide-sensitive factor; PAS: phagophore assembly site; PolyQ: polyglutamine; PtdIns3P: phosphatidylinositol-3-phosphate; PTK: protein tyrosine kinase; PTM: posttranslational modification; PTP: protein tyrosine phosphatase; PTPN23/HD-PTP: protein tyrosine phosphatase non-receptor type 23; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; STX7: syntaxin 7; STX8: syntaxin 8; STX17: syntaxin 17; VAMP3: vesicle associated membrane protein 3; VAMP7: vesicle associated membrane protein 7; VTI1B: vesicle transport through interaction with t-SNAREs 1B; YKT6: YKT6 v-SNARE homolog; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.
    Keywords:  ATG16l1; Autophagosome; PTPN9; SNARE; VTI1B
    DOI:  https://doi.org/10.1080/15548627.2020.1838117
  4. Front Cell Dev Biol. 2020 ;8 846
    Jiang Z, Zhang G, Huang L, Yuan Y, Wu C, Li Y.
      As the first compartment of the protein secretory pathway, the endoplasmic reticulum (ER) acts as a protein synthesis factory, maintaining proteostasis and ER homeostasis. However, a variety of intrinsic and extrinsic perturbations, such as cancer, can disrupt the homeostasis and result in a large accumulation of misfolded/unfolded proteins in the ER lumen, thereby provoking a specific cellular state addressed as "ER stress". Then the unfolded protein response (UPR), an adaptive signaling pathway, is triggered to address the stress and restore the homeostasis. A novel aspect of ER stress is that it can be transmitted from cancer cells to tumor-infiltrating myeloid cells through certain cancer cell-released soluble factors, which is termed as transmissible ER stress (TERS) or ER stress resonance (ERSR). In this review, we provide a comprehensive overview of the link between cancer and ER stress as well as the possible soluble factors mediating TERS. We further elaborate the cell-extrinsic effects of TERS on tumor immunity, and how it indirectly modulates cancer development and progression, which is expected to add a new dimension to anticancer therapy.
    Keywords:  cancer; transmissible ER stress; tumor immunity; tumor-derived extracellular vesicles; unfolded protein response
    DOI:  https://doi.org/10.3389/fcell.2020.00846
  5. Proc Natl Acad Sci U S A. 2020 Oct 26. pii: 202018578. [Epub ahead of print]
    Kober DL, Xu S, Li S, Bajaj B, Liang G, Rosenbaum DM, Radhakrishnan A.
      Lipid homeostasis in animal cells is maintained by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose proteolytic activation requires the cholesterol-sensing membrane protein Scap. In endoplasmic reticulum (ER) membranes, the carboxyl-terminal domain (CTD) of SREBPs binds to the CTD of Scap. When cholesterol levels are low, Scap escorts SREBPs from the ER to the Golgi, where the actions of two proteases release the amino-terminal domains of SREBPs that travel to the nucleus to up-regulate expression of lipogenic genes. The CTD of SREBP remains bound to Scap but must be eliminated so that Scap can be recycled to bind and transport additional SREBPs. Here, we provide insights into how this occurs by performing a detailed molecular dissection of the CTD of SREBP2, one of three SREBP isoforms expressed in mammals. We identify a degradation signal comprised of seven noncontiguous amino acids encoded in exon 19 that mediates SREBP2's proteasomal degradation in the absence of Scap. When bound to the CTD of Scap, this signal is masked and SREBP2 is stabilized. Binding to Scap requires an arginine residue in exon 18 of SREBP2. After SREBP2 is cleaved in Golgi, its CTD remains bound to Scap and returns to the ER with Scap where it is eliminated by proteasomal degradation. The Scap-binding motif, but not the degradation signal, is conserved in SREBP1. SREBP1's stability is determined by a degradation signal in a different region of its CTD. These findings highlight a previously unknown role for the CTD of SREBPs in regulating SREBP activity.
    Keywords:  Golgi; Scap; cholesterol; endoplasmic reticulum; proteasome
    DOI:  https://doi.org/10.1073/pnas.2018578117
  6. Biochem Soc Trans. 2020 Oct 30. 48(5): 2173-2184
    Chen YJ, Bagchi P, Tsai B.
      The endoplasmic reticulum (ER), with its expansive membranous system and a vast network of chaperones, enzymes, sensors, and ion channels, orchestrates diverse cellular functions, ranging from protein synthesis, folding, secretion, and degradation to lipid biogenesis and calcium homeostasis. Strikingly, some of the functions of the ER are exploited by viruses to promote their life cycles. During entry, viruses must penetrate a host membrane and reach an intracellular destination to express and replicate their genomes. These events lead to the assembly of new viral progenies that exit the host cell, thereby initiating further rounds of infection. In this review, we highlight how three distinct viruses - polyomavirus, flavivirus, and coronavirus - co-opt key functions of the ER to cause infection. We anticipate that illuminating this virus-ER interplay will provide rational therapeutic approaches to combat the virus-induced diseases.
    Keywords:  coronavirus; endoplasmic reticulum; flavivirus; polyomavirus; viral entry
    DOI:  https://doi.org/10.1042/BST20200395
  7. Proc Natl Acad Sci U S A. 2020 Oct 26. pii: 202014349. [Epub ahead of print]
    Zhang T, Periz G, Lu YN, Wang J.
      An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here, we report the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases.
    Keywords:  NEDD4L; SMAD; USP7; protein misfolding; protein quality control
    DOI:  https://doi.org/10.1073/pnas.2014349117
  8. Autophagy. 2020 Oct 29. 1-2
    Yang Y, Klionsky DJ.
      Reticulophagy, a type of selective autophagy that specifically targets and degrades parts of the endoplasmic reticulum (ER) network (sheets or tubules), plays a crucial role in the responses to ER stress. The selectivity of the ER cargo recognition relies on the unique reticulophagy receptors, which tether and deliver cargos to phagophores, the precursors to autophagosomes. Various integral membrane proteins have been well characterized as reticulophagy receptors, including Atg39, Atg40, RETREG1/FAM134B, SEC62, RTN3L, CCPG1, TEX264, and ATL3, in both yeast and mammals in the past five years. In a recent paper, Zhao et al. discovered in fission yeast a novel reticulophagy receptor, Epr1, which bridges the ER and phagophore by binding to Atg8 and VAPs, a mechanism different from the aforementioned reticulophagy receptors.
    Keywords:  Autophagy; endoplasmic reticulum; stress; vacuole; yeast
    DOI:  https://doi.org/10.1080/15548627.2020.1837457
  9. Planta. 2020 Oct 26. 252(5): 93
    Oh TR, Yu SG, Yang HW, Kim JH, Kim WT.
      MAIN CONCLUSION: AtKPNB1, an Arabidopsis importin-β protein, was regulated by AtAIRP1 E3 ubiquitin ligase, which intensified the ABA-mediated drought stress response. As an early step in the abscisic acid (ABA)-mediated drought response, the ABA signal is transduced into the nucleus, and thus the nuclear transport system is crucially involved in the drought stress response. AtKPNB1, an importin-β protein, which is a core component of nuclear transport, was previously reported to be a negative factor in the ABA-mediated drought stress response (Luo et al. Luo et al., Plant J 75:377-389, 2013). Here, we report that AtAIPR1, an Arabidopsis RING-type E3 ubiquitin (Ub) ligase, interacted with and ubiquitinated AtKPNB1. A null mutation of AtKPNB1 suppressed the ABA-insensitive germination phenotype of atairp1 mutant seedlings as compared to that of the wild-type plants. Furthermore, the ABA-insensitive stomatal closure and drought-susceptible phenotypes of atairp1 were rescued in atairp1atkpnb1 double mutant progeny, indicating that AtKPNB1 functions downstream of AtAIRP1. These data suggest that AtAIRP1 regulates the ABA-mediated drought response in Arabidopsis via ubiquitination of AtKPNB1.
    Keywords:  Abscisic acid; Arabidopsis; Drought stress response; Importin-β protein; RING-type E3 ubiquitin ligase; atairp1atkpnb1 double mutant
    DOI:  https://doi.org/10.1007/s00425-020-03500-4
  10. Cells. 2020 Oct 22. pii: E2339. [Epub ahead of print]9(11):
    Dastghaib S, Shojaei S, Mostafavi-Pour Z, Sharma P, Patterson JB, Samali A, Mokarram P, Ghavami S.
      Glioblastoma (GBM) is the most prevalent malignant primary brain tumor with a very poor survival rate. Temozolomide (TMZ) is the common chemotherapeutic agent used for GBM treatment. We recently demonstrated that simvastatin (Simva) increases TMZ-induced apoptosis via the inhibition of autophagic flux in GBM cells. Considering the role of the unfolded protein response (UPR) pathway in the regulation of autophagy, we investigated the involvement of UPR in Simva-TMZ-induced cell death by utilizing highly selective IRE1 RNase activity inhibitor MKC8866, PERK inhibitor GSK-2606414 (PERKi), and eIF2α inhibitor salubrinal. Simva-TMZ treatment decreased the viability of GBM cells and significantly increased apoptotic cell death when compared to TMZ or Simva alone. Simva-TMZ induced both UPR, as determined by an increase in GRP78, XBP splicing, eukaryote initiation factor 2α (eIF2α) phosphorylation, and inhibited autophagic flux (accumulation of LC3β-II and inhibition of p62 degradation). IRE1 RNase inhibition did not affect Simva-TMZ-induced cell death, but it significantly induced p62 degradation and increased the microtubule-associated proteins light chain 3 (LC3)β-II/LC3β-I ratio in U87 cells, while salubrinal did not affect the Simva-TMZ induced cytotoxicity of GBM cells. In contrast, protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibition significantly increased Simva-TMZ-induced cell death in U87 cells. Interestingly, whereas PERK inhibition induced p62 accumulation in both GBM cell lines, it differentially affected the LC3β-II/LC3β-I ratio in U87 (decrease) and U251 (increase) cells. Simvastatin sensitizes GBM cells to TMZ-induced cell death via a mechanism that involves autophagy and UPR pathways. More specifically, our results imply that the IRE1 and PERK signaling arms of the UPR regulate Simva-TMZ-mediated autophagy flux inhibition in U251 and U87 GBM cells.
    Keywords:  ER stress; autophagy; autophagy flux; glioblastoma; mevalonate cascade; statin
    DOI:  https://doi.org/10.3390/cells9112339
  11. EMBO J. 2020 Oct 30. e104369
    Di Mattia T, Martinet A, Ikhlef S, McEwen AG, Nominé Y, Wendling C, Poussin-Courmontagne P, Voilquin L, Eberling P, Ruffenach F, Cavarelli J, Slee J, Levine TP, Drin G, Tomasetto C, Alpy F.
      Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.
    Keywords:  cholesterol; inter-organelle contact; lipid transfer protein; regulation; small linear motif
    DOI:  https://doi.org/10.15252/embj.2019104369
  12. Cells. 2020 Oct 24. pii: E2349. [Epub ahead of print]9(11):
    Vainshtein A, Grumati P.
      Autophagy, a bulk degradation process within eukaryotic cells, is responsible for cellular turnover and nutrient liberation during starvation. Increasing evidence indicate that this process can be extremely discerning. Selective autophagy segregates and eliminates protein aggregates, damaged organelles, and invading organisms. The specificity of this process is largely mediated by post-translational modifications (PTMs), which are recognized by autophagy receptors. These receptors grant autophagy surgical precision in cargo selection, where only tagged substrates are engulfed within autophagosomes and delivered to the lysosome for proteolytic breakdown. A growing number of selective autophagy receptors have emerged including p62, NBR1, OPTN, NDP52, TAX1BP1, TOLLIP, and more continue to be uncovered. The most well-documented PTM is ubiquitination and selective autophagy receptors are equipped with a ubiquitin binding domain and an LC3 interacting region which allows them to physically bridge cargo to autophagosomes. Here, we review the role of ubiquitin and ubiquitin-like post-translational modifications in various types of selective autophagy.
    Keywords:  ER-phagy; aggrephagy; cargo receptors; lipophagy; lysophagy; mitophagy; nucleophagy; selective autophagy; ubiquitin; xenophagy
    DOI:  https://doi.org/10.3390/cells9112349
  13. Life Sci Alliance. 2020 Dec;pii: e202000815. [Epub ahead of print]3(12):
    Zachari M, Longo M, Ganley IG.
      Autophagy is a crucial homeostatic mechanism that mediates the degradation of damaged or excess intracellular components. Such components are engulfed and sequestered into double membrane autophagosomes, which deliver their contents to lysosomes for degradation. Autophagy plays a role in numerous human disorders and its pharmacological targeting by small molecules offers therapeutic potential. The serine/threonine kinase ULK1 (and its homologue ULK2) is the most upstream component of the autophagic machinery and is required for autophagy initiation. Here, we use the most selective and potent published ULK1 inhibitors to gain insights into ULK1 kinase function during autophagy. Treatment with all inhibitors blocked autophagy but also resulted in the limited formation of initial autophagosome-like structures, which appeared abnormal in size and did not traffic to lysosomes. We found that upon ULK1 inhibition, phosphatidylinositol-3-phosphate-binding proteins are still recruited to forming autophagosomes, implying that ULK1 activity is not essential for VPS34 activation. We conclude that the kinase activity of ULK1 is important in regulating autophagosome maturation, by the phosphorylation of currently unidentified key substrates.
    DOI:  https://doi.org/10.26508/lsa.202000815
  14. iScience. 2020 Oct 23. 23(10): 101648
    Miyata T, Hagiwara D, Hodai Y, Miwata T, Kawaguchi Y, Kurimoto J, Ozaki H, Mitsumoto K, Takagi H, Suga H, Kobayashi T, Sugiyama M, Onoue T, Ito Y, Iwama S, Banno R, Matsumoto M, Kawakami N, Ohno N, Sakamoto H, Arima H.
      Misfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.
    Keywords:  cell biology; neuroscience; technical aspects of cell biology
    DOI:  https://doi.org/10.1016/j.isci.2020.101648
  15. J Mol Cell Cardiol. 2020 Oct 21. pii: S0022-2828(20)30305-9. [Epub ahead of print]
    Wang N, Ma H, Li J, Meng C, Zou J, Wang H, Liu K, Liu M, Xiao X, Zhang H, Wang K.
      Palmitic acid (PA)-induced myocardial injury is considered a critical contributor to the development of obesity and type 2 diabetes mellitus (T2DM)-related cardiomyopathy. However, the underlying mechanism has not been fully understood. Here, we demonstrated that PA induced the cell death of H9c2 cardiomyoblasts in a dose- and time-dependent manner, while different ferroptosis inhibitors significantly abrogated the cell death of H9c2 cardiomyoblasts and primary neonatal rat cardiomyocytes exposed to PA. Mechanistically, PA decreased the protein expression levels of both heat shock factor 1 (HSF1) and glutathione peroxidase 4 (GPX4) in a dose- and time-dependent manner, which were restored by different ferroptosis inhibitors. Overexpression of HSF1 not only alleviated PA-induced cell death and lipid peroxidation but also improved disturbed iron homeostasis by regulating the transcription of iron metabolism-related genes (e.g., Fth1, Tfrc, Slc40a1). Additionally, PA-blocked GPX4 protein expression was evidently restored by HSF1 overexpression. Inhibition of endoplasmic reticulum (ER) stress rather than autophagy contributed to HSF1-mediated GPX4 expression. Moreover, GPX4 overexpression protected against PA-induced ferroptosis, whereas knockdown of GPX4 reversed the anti-ferroptotic effect of HSF1. Consistent with the in vitro findings, PA-challenged Hsf1-/- mice exhibited more serious ferroptosis, increased Slc40a1 and Fth1 mRNA expression, decreased GPX4 and TFRC expression and enhanced ER stress in the heart compared with Hsf1+/+ mice. Altogether, HSF1 may function as a key defender against PA-induced ferroptosis in cardiomyocytes by maintaining cellular iron homeostasis and GPX4 expression.
    Keywords:  Cardiomyocyte; Endoplasmic reticulum stress; Ferroptosis; Heat shock factor 1; Palmitic acid
    DOI:  https://doi.org/10.1016/j.yjmcc.2020.10.010
  16. J Cell Sci. 2020 Oct 26. pii: jcs.252015. [Epub ahead of print]
    Corkery DP, Nadeem A, Aung KM, Hassan A, Liu T, Cervantes-Rivera R, Lystad AH, Wang H, Persson K, Puhar A, Simonsen A, Uhlin BE, Wai SN, Wu YW.
      Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.
    Keywords:  Bacterial toxin; IL-1 beta; MakA; Membrane aggregate; Noncanonical autophagy; Unconventional secretion
    DOI:  https://doi.org/10.1242/jcs.252015
  17. Nat Struct Mol Biol. 2020 Oct 26.
    Matoba K, Kotani T, Tsutsumi A, Tsuji T, Mori T, Noshiro D, Sugita Y, Nomura N, Iwata S, Ohsumi Y, Fujimoto T, Nakatogawa H, Kikkawa M, Noda NN.
      The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
    DOI:  https://doi.org/10.1038/s41594-020-00518-w
  18. Cell Stress Chaperones. 2020 Oct 29.
    Bressler KR, Ross JA, Ilnytskyy S, Vanden Dungen K, Taylor K, Patel K, Zovoilis A, Kovalchuk I, Thakor N.
      During the integrated stress response (ISR), global translation initiation is attenuated; however, noncanonical mechanisms allow for the continued translation of specific transcripts. Eukaryotic initiation factor 5B (eIF5B) has been shown to play a critical role in canonical translation as well as in noncanonical mechanisms involving internal ribosome entry site (IRES) and upstream open reading frame (uORF) elements. The uORF-mediated translation regulation of activating transcription factor 4 (ATF4) mRNA plays a pivotal role in the cellular ISR. Our recent study confirmed that eIF5B depletion removes uORF2-mediated repression of ATF4 translation, which results in the upregulation of growth arrest and DNA damage-inducible protein 34 (GADD34) transcription. Accordingly, we hypothesized that eIF5B depletion may reprogram the transcriptome profile of the cell. Here, we employed genome-wide transcriptional analysis on eIF5B-depleted cells. Further, we validate the up- and downregulation of several transcripts from our RNA-seq data using RT-qPCR. We identified upregulated pathways including cellular response to endoplasmic reticulum (ER) stress, and mucin-type O-glycan biosynthesis, as well as downregulated pathways of transcriptional misregulation in cancer and T cell receptor signaling. We also confirm that depletion of eIF5B leads to activation of the c-Jun N-terminal kinase (JNK) arm of the mitogen-activated protein kinase (MAPK) pathway. This data suggests that depletion of eIF5B reprograms the cellular transcriptome and influences critical cellular processes such as ER stress and ISR.
    Keywords:  ATF4; ER stress; Eukaryotic initiation factor 5B (eIF5B); ISR; JNK; Transcriptome
    DOI:  https://doi.org/10.1007/s12192-020-01174-1
  19. FEBS J. 2020 Oct 30.
    Bchini R, Girardet JM, Sormani R, Gelhaye E, Morel-Rouhier M.
      The Eukaryotic Translation Elongation Factor 1Bγ (eEF1Bγ) is an atypical member of the glutathione transferase (GST) superfamily. Contrary to more classical GSTs having a role in toxic compound detoxification, eEF1Bγ is suggested to act as a scaffold protein, anchoring the elongation factor complex EF1B to the endoplasmic reticulum. In this study we show that eEF1Bγ from the basidiomycete Phanerochaete chrysosporium is fully active as a glutathione transferase in vitro and undergoes conformational changes upon binding of oxidized glutathione. Using real-time analyses of biomolecular interactions, we show that GSSG allows eEF1Bγ to physically interact with other GSTs from the Ure2p class, opening new perspectives for a better understanding of the role of eEF1Bγ in cellular oxidative stress response.
    Keywords:  GSSG; eEF1Bγ; glutathione transferase; time-resolved molecular dynamics
    DOI:  https://doi.org/10.1111/febs.15614
  20. FEBS J. 2020 Oct 28.
    Klimpel A, Stillger K, Wiederstein JL, Krüger M, Neundorf I.
      Cysteine prenylation is a post-translational modification that is used by nature to control crucial biological functions of proteins, such as membrane trafficking, signal transduction and apoptosis. It mainly occurs in eukaryotic proteins at a C-terminal CaaX-box and is mediated by prenyltransferases. Since the discovery of prenylated proteins various tools have been developed to study the mechanisms of prenyltransferases, as well as to visualize and to identify prenylated proteins. Herein, we introduce cell-permeable peptides bearing a C-terminal CaaX-motif based on Ras sequences. We demonstrate that intracellular accumulation of those peptides in different cells is controlled by the presence of their CaaX-motif, and that they specifically interact with intracellular prenyltransferases. As proof of concept, we further highlight their utilization to alter downstream signaling of Ras proteins, particularly of K-Ras-4B, in pancreatic cancer cells. Application of this strategy holds great promise to better understand and regulate post-translational cysteine prenylation.
    Keywords:  CaaX-motif; Ras-proteins; cell-penetrating peptides; cysteine prenylation; farnesyltransferase
    DOI:  https://doi.org/10.1111/febs.15612
  21. Elife. 2020 Oct 28. pii: e53734. [Epub ahead of print]9
    Li XL, Pongor L, Tang W, Das S, Muys BR, Jones MF, Lazar SB, Dangelmaier EA, Hartford CC, Grammatikakis I, Hao Q, Sun Q, Schetter A, Martindale JL, Tang B, Jenkins LM, Robles AI, Walker RL, Ambs S, Chari R, Shabalina SA, Gorospe M, Hussain PS, Harris CC, Meltzer PS, Prasanth KV, Aladjem MI, Andresson T, Lal A.
      Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal tract-specific lncRNA (LINC00675) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP (FOXA1-Regulated Conserved Small Protein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells.
    Keywords:  cancer biology; chromosomes; gene expression; human
    DOI:  https://doi.org/10.7554/eLife.53734
  22. Mol Cell. 2020 Oct 20. pii: S1097-2765(20)30690-0. [Epub ahead of print]
    Lin J, Chen K, Chen W, Yao Y, Ni S, Ye M, Zhuang G, Hu M, Gao J, Gao C, Liu Y, Yang M, Zhang Z, Zhang X, Huang J, Chen F, Sun L, Zhang X, Yu S, Chen Y, Jiang Y, Wang S, Yang X, Liu K, Zhou HM, Ji Z, Deng H, Haque ME, Li J, Mi LZ, Li Y, Yang Y.
      Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.
    Keywords:  Atg5-Atg12; PINK1; Parkin; Parkinson’s disease; TUFm; homeostatic control; mitophagy; paradoxical signaling; robustness; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2020.10.007
  23. Oncol Rep. 2020 Oct 20.
    Alhammad R, Khunchai S, Tongmuang N, Limjindaporn T, Yenchitsomanus PT, Mutti L, Krstic-Demonacos M, Demonacos C.
      Oxidoreductase protein disulphide isomerases (PDI) are involved in the regulation of a variety of biological processes including the modulation of endoplasmic reticulum (ER) stress, unfolded protein response (UPR), ER‑mitochondria communication and the balance between pro‑survival and pro‑death pathways. In the current study the role of the PDIA1 family member in breast carcinogenesis was investigated by measuring ROS generation, mitochondrial membrane disruption, ATP production and HLA‑G protein levels on the surface of the cellular membrane in the presence or absence of PDIA1. The results showed that this enzyme exerted pro‑apoptotic effects in estrogen receptor (ERα)‑positive breast cancer MCF‑7 and pro‑survival in triple negative breast cancer (TNBC) MDA‑MB‑231 cells. ATP generation was upregulated in PDIA1‑silenced MCF‑7 cells and downregulated in PDIA1‑silenced MDA‑MB‑231 cells in a manner dependent on the cellular redox status. Furthermore, MCF‑7 and MDA‑MB‑231 cells in the presence of PDIA1 expressed higher surface levels of the non‑classical human leukocyte antigen (HLA‑G) under oxidative stress conditions. Evaluation of the METABRIC datasets showed that low PDIA1 and high HLA‑G mRNA expression levels correlated with longer survival in both ERα‑positive and ERα‑negative stage 2 breast cancer patients. In addition, analysis of the PDIA1 vs. the HLA‑G mRNA ratio in the subgroup of the living stage 2 breast cancer patients exhibiting low PDIA1 and high HLA‑G mRNA levels revealed that the longer the survival time of the ratio was high PDIA1 and low HLA‑G mRNA and occurred predominantly in ERα‑positive breast cancer patients whereas in the same subgroup of the ERα‑negative breast cancer mainly this ratio was low PDIA1 and high HLA‑G mRNA. Taken together these results provide evidence supporting the view that PDIA1 is linked to several hallmarks of breast cancer pathways including the process of antigen processing and presentation and tumor immunorecognition.
    DOI:  https://doi.org/10.3892/or.2020.7816
  24. J Lipid Res. 2020 Oct 27. pii: jlr.RA120001006. [Epub ahead of print]
    De Giorgi M, Jarrett KE, Burton JC, Doerfler AM, Hurley A, Li A, Hsu RH, Furgurson M, Patel KR, Han J, Borchers CH, Lagor WR.
      HMG-CoA Reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing non-sterol isoprenoids, such as dolichol, ubiquinone, farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on non-sterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the non-sterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr null hepatocytes showed endoplasmic reticulum (ER) stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress.  Using AAV-CRISPR, we somatically disrupted Dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver, and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.
    Keywords:  AAV; CRISPR/Cas9; Cholesterol/Biosynthesis; Dhdds; Dolichol; Endoplasmic reticulum stress; Hmgcr; Isoprenoids; Liver; Sterols
    DOI:  https://doi.org/10.1194/jlr.RA120001006
  25. Autophagy. 2020 Oct 28.
    Zou B, Liu J, Klionsky DJ, Tang D, Kang R.
      Excessive inflammation may lead to irreparable injury and even death, but the key mediators and underlying mechanisms remain unclear. Our recent findings indicate that SQSTM1/p62 (sequestosome 1), a well-known macroautophagy/autophagy receptor, is a lethal inflammatory mediator of sepsis and septic shock. The release of SQSTM1 occurs during tissue damage or microbial invasion through two main ways: one is passive and the other is active. Passive release occurs in the context of GSDMD-mediated pyroptosis. Active SQSTM1 secretion requires two basic steps: the first step is the expression and phosphorylation of SQSTM1 mediated by STING1/STING/TMEM173, and then the unconventional secretion of SQSTM1 by secretory lysosomes. After release, the extracellular SQSTM1 binds to membrane receptor INSR to activate glycolysis, leading to subsequent production of pro-inflammatory cytokines in a transcription factor NFKB-dependent manner. Functionally, genetic deletion or pharmacological inhibition of the SQSTM1-INSR pathway limits tissue damage, systemic inflammation, organ failure, and death in experimental sepsis models in mice. Moreover, the activation of the SQSTM1-INSR pathway is related to the severity of sepsis in patients. These findings highlight a pathological role of extracellular SQSTM1 in infection, inflammation, and immunity.
    Keywords:  DAMP; INSR; SQSTM1; STING1; TLR4; autophagy; immunometabolism; inflammasome; sepsis
    DOI:  https://doi.org/10.1080/15548627.2020.1843253
  26. Chemistry. 2020 Oct 26.
    Ito Y, Kajihara Y, Takeda Y.
      The introduction of Asn-linked glycans to nascent polypeptides occurs in the lumen of the endoplasmic reticulum of eukaryotic cells. After the removal of specific sugar residues, glycoproteins acquire signals in the glycoprotein quality control (GPQC) system and enter the folding cycle composed of lectin-chaperones calnexin (CNX) and calreticulin (CRT), glucosidase II (G-II), and UDP-Glc:glycoprotein glucosyltransferase (UGGT). G-II initiates glycoproteins' entry and exit from the cycle, and UGGT serves as the "folding sensor".
    Keywords:  Chemical synthesis of glycoproteins; Glycoproteins; Quality control; Stereoselective glycosylation; The endoplasmic reticulum
    DOI:  https://doi.org/10.1002/chem.202004158
  27. Cancer Immunol Immunother. 2020 Oct 26.
    Andrews AM, Tennant MD, Thaxton JE.
      The solid tumor microenvironment is replete with factors that present a stress to infiltrating immune cells. Endoplasmic reticulum (ER) stress sensor PKR-like ER kinase (PERK) is primed to sense and respond to the burden of misfolded proteins in the ER lumen induced by cell stressors. PERK has documented roles as a master regulator of acute and chronic responses to cell stress as well as in the regulation of cell metabolism. Here, we provide an overview of the roles of PERK based on what is known and remains to be tested in immune cells in tumors and impacts on tumor control. PERK is one of several ER kinases able to preferentially induce activating transcription factor 4 (ATF4) as a response to cell stress. ATF4 orchestrates the oxidative stress response and governs amino acid metabolism. We discuss the tested role of ATF4 in tumor immunity and provide insight on the dueling protective and deleterious roles that ATF4 may play in the stress of solid tumors.
    Keywords:  ATF4; Cancer immunotherapy; ER stress; Metabolism; PERK; T cell; Translation
    DOI:  https://doi.org/10.1007/s00262-020-02740-3
  28. Sci Rep. 2020 Oct 28. 10(1): 18419
    Kunz V, Bommert KS, Kruk J, Schwinning D, Chatterjee M, Stühmer T, Bargou R, Bommert K.
      Experimental evidence suggests that ubiquitin-protein ligases regulate a number of cellular processes involved in tumorigenesis. We analysed the role of the E3 ubiquitin-protein ligase HUWE1 for pathobiology of multiple myeloma (MM), a still incurable blood cancer. mRNA expression analysis indicates an increase in HUWE1 expression levels correlated with advanced stages of myeloma. Pharmacologic as well as RNAi-mediated HUWE1 inhibition caused anti-proliferative effects in MM cell lines in vitro and in an MM1.S xenotransplantation mouse model. Cell cycle analysis upon HUWE1 inhibition revealed decreased S phase cell fractions. Analyses of potential HUWE1-dependent molecular functions did not show involvement in MYC-dependent gene regulation. However, HUWE1 depleted MM cells displayed increased DNA tail length by comet assay, as well as changes in the levels of DNA damage response mediators such as pBRCA1, DNA-polymerase β, γH2AX and Mcl-1. Our finding that HUWE1 might thus be involved in endogenous DNA repair is further supported by strongly enhanced apoptotic effects of the DNA-damaging agent melphalan in HUWE1 depleted cells in vitro and in vivo. These data suggest that HUWE1 might contribute to tumour growth by endogenous repair of DNA, and could therefore potentially be exploitable in future treatment developments.
    DOI:  https://doi.org/10.1038/s41598-020-75499-3
  29. Cells. 2020 Oct 26. pii: E2359. [Epub ahead of print]9(11):
    Filippopoulou C, Simos G, Chachami G.
      Sumoylation is the covalent attachment of the small ubiquitin-related modifier (SUMO) to a vast variety of proteins in order to modulate their function. Sumoylation has emerged as an important modification with a regulatory role in the cellular response to different types of stress including osmotic, hypoxic and oxidative stress. Hypoxia can occur under physiological or pathological conditions, such as ischemia and cancer, as a result of an oxygen imbalance caused by low supply and/or increased consumption. The hypoxia inducible factors (HIFs), and the proteins that regulate their fate, are critical molecular mediators of the response to hypoxia and modulate procedures such as glucose and lipid metabolism, angiogenesis, erythropoiesis and, in the case of cancer, tumor progression and metastasis. Here, we provide an overview of the sumoylation-dependent mechanisms that are activated under hypoxia and the way they influence key players of the hypoxic response pathway. As hypoxia is a hallmark of many diseases, understanding the interrelated connections between the SUMO and the hypoxic signaling pathways can open the way for future molecular therapeutic interventions.
    Keywords:  HIF; HIF-1α; SUMO; hypoxia; oxygen homeostasis; sumoylation
    DOI:  https://doi.org/10.3390/cells9112359
  30. Cell Death Differ. 2020 Oct 25.
    Chen X, Guan Y, Zhang Y, Jia Y, Li W, Guo C, Li Y, Wang X, Shi Y, Wang Q, Zhu F, Li Y, Zhang L.
      Transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis. The post-translational phosphorylation modulations of TFEB by mTOR and ERK signaling can determine its nucleocytoplasmic shuttling and activity in response to nutrient availability. However, regulations of TFEB at translational level are rarely known. Here, we found that programmed cell death 4 (PDCD4), a tumor suppressor, decreased levels of nuclear TFEB to inhibit lysosome biogenesis and function. Mechanistically, PDCD4 reduces global pool of TFEB by suppressing TFEB translation in an eIF4A-dependent manner, rather than influencing mTOR- and ERK2-dependnet TFEB nucleocytoplasmic shuttling. Both of MA3 domains within PDCD4 are required for TFEB translation inhibition. Furthermore, TFEB is required for PDCD4-mediated lysosomal function suppression. In the tumor microenvironment, PDCD4 deficiency promotes the anti-tumor effect of macrophage via enhancing TFEB expression. Our research reveals a novel PDCD4-dependent TFEB translational regulation and supports PDCD4 as a potential therapeutic target for lysosome dysfunction related diseases.
    DOI:  https://doi.org/10.1038/s41418-020-00646-2
  31. J Immunol. 2020 Oct 30. pii: ji1901384. [Epub ahead of print]
    Peng Y, Guo J, Sun T, Fu Y, Zheng H, Dong C, Xiong S.
      Deubiquitinating enzymes (DUBs) are cysteine proteases that reverse the ubiquitination by removing ubiquitins from the target protein. The human genome encodes ∼100 potential DUBs, which can be classified into six families, influencing multiple cellular processes, such as antiviral responses, inflammatory responses, apoptosis, etc. To systematically explore the role of DUBs involved in antiviral immunity, we performed an RNA interference-based screening that contains 97 human DUBs. We identified that ubiquitin-specific protease (USP) 39 expression modulates the antiviral activity, which is, to our knowledge, a previously unknown function of this enzyme. Small interfering RNA knockdown of USP39 significantly enhanced viral replication, whereas overexpression of USP39 had an opposite effect. Mechanistically, USP39 does not affect the production of type I IFN but significantly promotes JAK/STAT downstream of type I signaling by enhancing IFN-stimulated response elements promoter activity and expression of IFN-stimulated genes. Interestingly, USP39, previously considered not to have the deubiquitinase activity, in this study is proved to interact with STAT1 and sustain its protein level by deubiqutination. Furthermore, we found that through novel mechanism USP39 can significantly decrease K6-linked but not K48-linked ubiquitination of STAT1 for degradation. Taken together, these findings uncover that USP39 is, to our knowledge, a new deubiquitinase that positively regulates IFN-induced antiviral efficacy.
    DOI:  https://doi.org/10.4049/jimmunol.1901384
  32. Biochem Biophys Rep. 2020 Dec;24 100834
    Nomura Y, Sylvester CF, Nguyen LO, Kandeel M, Hirata Y, Mungrue IN, Oh-Hashi K.
      The Unfolded Protein Response pathway is a conserved signaling mechanism having important roles in cellular physiology and is perturbed accompanying disease. We previously identified the novel UPR target gene CHAC1, a direct target of ATF4, downstream of PERK-EIF2A and activated by the UPR pathway. CHAC1 enzyme directs catalysis of γ-linked glutamate bonds within specific molecular targets. CHAC1 is the first enzyme characterized that can catalyze intracellular glutathione degradation in eukaryotes, having implications for regulation of oxidative stress. DDIT3 (CHOP) is a terminal UPR transcription factor, regulated by ATF4 and an output promoting cell death signaling. Herein we examine the relationship of CHOP controlling CHAC1 transcription in humans and mice. We note parallel induction of CHOP and CHAC1 in human cells after agonist induced UPR. Expanding upon previous reports, we define transcriptional induction of CHAC1 in humans and mice driven by ATF4 through a synergistic relationship with conserved ATF/CRE and CARE DNA sequences of the CHAC1 promoter. Using this system, we also tested effects of CHOP on CHAC1 transcription, and binding at the CHAC1 ATF/CRE using IM-EMSA. These data indicate a novel inhibitory effect of CHOP on CHAC1 transcription, which was ablated in the absence of the ATF/CRE control element. While direct binding of ATF4 to CHAC1 promoter sequences was confirmed, binding of CHOP to the CHAC1 ATF/CRE was not evident at baseline or after UPR induction. These data reveal CHAC1 as a novel CHOP inhibited target gene, acting through an upstream ATF/CRE motif via an indirect mechanism.
    Keywords:  ATF/CRE; ATF4; CHAC1; DDIT3
    DOI:  https://doi.org/10.1016/j.bbrep.2020.100834
  33. Cancers (Basel). 2020 Oct 23. pii: E3093. [Epub ahead of print]12(11):
    Rodríguez-Alonso A, Casas-Pais A, Roca-Lema D, Graña B, Romay G, Figueroa A.
      The epithelial-mesenchymal plasticity (EMP) is a process by which epithelial cells acquire the ability to dynamically switch between epithelial and mesenchymal phenotypic cellular states. Epithelial cell plasticity in the context of an epithelial-to-mesenchymal transition (EMT) confers increased cell motility, invasiveness and the ability to disseminate to distant sites and form metastasis. The modulation of molecularly defined targets involved in this process has become an attractive therapeutic strategy against cancer. Protein degradation carried out by ubiquitination has gained attention as it can selectively degrade proteins of interest. In the ubiquitination reaction, the E3 ubiquitin-ligases are responsible for the specific binding of ubiquitin to a small subset of target proteins, and are considered promising anticancer drug targets. In this review, we summarize the role of the E3 ubiquitin-ligases that control targeted protein degradation in cancer-EMT, and we highlight the potential use of the E3 ubiquitin-ligases as drug targets for the development of small-molecule drugs against cancer.
    Keywords:  E3 ubiquitin-ligases; cancer; drug targets; epithelial–mesenchymal plasticity; posttranslational regulation; targeted protein degradation; ubiquitination
    DOI:  https://doi.org/10.3390/cancers12113093
  34. Mol Cell. 2020 Oct 15. pii: S1097-2765(20)30686-9. [Epub ahead of print]
    Lees JA, Li P, Kumar N, Weisman LS, Reinisch KM.
      The phosphoinositide PI(3,5)P2, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P2 levels within cells are regulated. We find the PIKfyve complex comprises five copies of the scaffolding protein Vac14 and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P2 from PI3P and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P2 production by PIKfyve in vivo.
    Keywords:  lipid kinase; lipid phosphatase; phosphoinositide homeostasis
    DOI:  https://doi.org/10.1016/j.molcel.2020.10.003
  35. PLoS Genet. 2020 Oct 28. 16(10): e1009091
    Ishikawa K, Ishihara A, Moriya H.
      Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 12 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent stoichiometry control. Our study reveals the complexity and robustness of the stoichiometry control system.
    DOI:  https://doi.org/10.1371/journal.pgen.1009091
  36. Oncogenesis. 2020 Oct 26. 9(10): 96
    García-Heredia JM, Otero-Albiol D, Pérez M, Pérez-Castejón E, Muñoz-Galván S, Carnero A.
      MAP17 (PDZK1IP1) is a small protein regulating inflammation and tumor progression, upregulated in a broad range of carcinomas. MAP17 levels increase during tumor progression in a large percentage of advanced tumors. In the present work, we explored the role of this protein shaping tumor evolution. Here we show that in breast cancer, cells increased MAP17 levels in tumors by demethylation induced multiple changes in gene expression through specific miRNAs downregulation. These miRNA changes are dependent on Notch pathway activation. As a consequence, epithelial mesenchymal transition (EMT) and stemness are induced promoting the metastatic potential of these cells both in vitro and in vivo. Furthermore, MAP17 increased the exosomes in tumor cells, where MAP17 was released as cargo, and this horizontal propagation also increased the EMT in the recipient cells. Importantly, an antibody against MAP17 in the media reduces the EMT and stemness alterations promoted by the conditioned media from MAP17-expressing cells. Therefore, MAP17 expression promotes the horizontal propagation of EMT and metastasis by transferring the MAP17 protein between subsets of neoplastic cells. Thus, MAP17 can be used to describe a new mechanism for cell malignity at distance, without the involvement of genetic or epigenetic modifications. MAP17 can also be taken in consideration as new target for metastatic high-grade breast tumors.
    DOI:  https://doi.org/10.1038/s41389-020-00280-0
  37. PLoS Pathog. 2020 Oct 27. 16(10): e1008784
    Burge RJ, Damianou A, Wilkinson AJ, Rodenko B, Mottram JC.
      Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.
    DOI:  https://doi.org/10.1371/journal.ppat.1008784
  38. Free Radic Biol Med. 2020 Oct 27. pii: S0891-5849(20)31302-2. [Epub ahead of print]
    Wang P, Yang Y, Pang G, Zhang C, Wei C, Tao X, Liu J, Xu J, Zhang W, Shen Y.
      Rifampicin (RFP) has been known to be potentially hepatotoxic and often used as an inducer of cholestatic hepatic injury. Here we found that mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein, is a protector in RFP-induced liver injury. In cholestatic hepatic injury mice induced by RFP, the liver/body ratio and the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) were significantly increased. Meanwhile, the protein and mRNA levels of MANF were remarkably elevated in the liver injury mice. In hepatocyte-specific MANF knockout (HKO) mice, an extra increase in the liver/body ratio and serum ALT, AST, ALP, TBA, TBIL, and DBIL levels was detected after treatment with RFP. In addition, recombinant human MANF (rhMANF) treatment efficiently reduced the liver/body ratio and serum ALT, AST, ALP, TBA, TBIL, and DBIL levels in RFP-induced liver injury mice. Furthermore, we found there is an increase in the number of the apoptotic cells, detected by TUNEL staining in the liver tissues of HKO mice. Meanwhile, the protein levels of C/EBP-homologous protein (CHOP), Ki67, and the proliferating cell nuclear antigen (PCNA), as well as the mRNA level of Ki67 were elevated after treated with RFP, and these parameters were increased more significantly in HKO mice than that in wild type (WT) controls in RFP-induced liver injury. The rhMANF treatment can rescue the cell apoptosis and reduce the protein and mRNA levels of CHOP, Ki67, and PCNA elevated by MANF deletion and RFP. In HKO mice, immunoglobulin heavy chain binding protein (BIP) and activating transcription factor 4 (ATF4) were predominantly increased after treatment with RFP, which were reduced by rhMANF treatment. Therefore, we conclude that hepatocyte-derived MANF is protective for RFP-induced cholestatic hepatic injury via inhibiting ATF4-CHOP signal activation and subsequent cell apoptosis.
    Keywords:  ATF4; ER stress; MANF; rifampicin-induced cholestatic hepatic injury
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2020.10.028
  39. Dis Model Mech. 2020 Oct 08. pii: dmm.041350. [Epub ahead of print]
    Doyle JJ, Vrancx C, Maios C, Labarre A, Patten SA, Parker JA.
      Spinal muscular atrophy is (SMA) is a devastating, autosomal recessive neuromuscular disease resulting in muscle atrophy, neurodegeneration, and is the leading genetic cause of infant death. SMA arises when there are homozygous deletion mutations in the human SMN1 gene, leading to a decrease in corresponding SMN1 protein. Although SMN1 is expressed across multiple tissue types, much of the previous research into SMA focused on the neuronal aspect of the disease, overlooking many of the potential non-neuronal aspects of the disease. Therefore, we sought to address this gap in knowledge by modeling SMA in the nematode Caenorhabditis elegans We used a previously uncharacterized allele which resulted in the onset of mild SMA-like phenotypes allowing us to monitor the onset of phenotypes at different stages. We observed that these mutant animals recapitulated many key features of the human disease, and most importantly, we observed that muscle dysfunction precedes neurodegeneration. Furthermore, we tested the therapeutic efficacy of targeting endoplasmic reticulum (ER) stress in non-neuronal cells and found it to be more effective than targeting ER stress in neuronal cells. We also found that the most potent therapeutic potential came from a combination of ER- and neuromuscular junction (NMJ)-targeting drugs. Together, our results suggest an important non-neuronal component of SMA pathology and highlight new considerations for therapeutic intervention.
    Keywords:  C. elegans; ER stress; Genetics; Muscle pathology; Spinal muscular atrophy
    DOI:  https://doi.org/10.1242/dmm.041350
  40. Plant J. 2020 Oct 24.
    Ko DK, Brandizzi F.
      Adverse environmental conditions reduce crop productivity and often increase a load of unfolded or misfolded proteins in the endoplasmic reticulum (ER). This potentially lethal condition, known as ER stress, is buffered by the unfolded protein response (UPR), a set of signaling pathways designed to either recover ER functionality or ignite programmed cell death. Despite the biological significance of the UPR to the life of the organism, the regulatory transcriptional landscape underpinning ER stress management is largely unmapped, especially in crops. To fill this significant knowledge gap, we performed a large-scale systems-level analysis of protein-DNA interaction (PDI) network in maize (Zea mays). Using 23 promoter fragments of six UPR marker genes in a high-throughput enhanced yeast one-hybrid (eY1H) assay, we identified a highly interconnected network of 262 transcription factors (TFs) associated with significant biological traits and 831 PDIs underlying the UPR. We established a temporal hierarchy of TF binding to gene promoters within the same family as well as across different families of TFs. Cistrome analysis revealed the dynamic activities of a variety of cis-regulatory elements (CREs) in ER stress-responsive gene promoters. By integrating the cistrome results into a TF network analysis, we mapped a subnetwork of TFs associated with a CRE that may contribute to the UPR management. Finally, we validated the role of a predicted network hub gene using the Arabidopsis system. The PDIs, TF networks and CREs identified in our work are foundational resources for understanding transcription regulatory mechanisms in the stress responses and crop improvement.
    Keywords:  ER stress; TF network; Transcription factors; UPR; cistrome; gene regulation; protein-DNA interaction; yeast one-hybrid
    DOI:  https://doi.org/10.1111/tpj.15044
  41. Elife. 2020 10 27. pii: e55694. [Epub ahead of print]9
    Nuckolls NL, Mok AC, Lange JJ, Yi K, Kandola TS, Hunn AM, McCroskey S, Snyder JL, Bravo Núñez MA, McClain M, McKinney SA, Wood C, Halfmann R, Zanders SE.
      Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism to selectively kill meiotic products (spores) that do not inherit wtf4. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate trafficking, as mutations that disrupt either of these processes result in cell death in the presence of the Wtf4 proteins. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy spores.
    Keywords:  Meiotic drive; S. cerevisiae; S. pombe; autophagy; cell biology; evolutionary biology; meiosis; protein aggregation; proteostasis; wtf
    DOI:  https://doi.org/10.7554/eLife.55694
  42. Adv Sci (Weinh). 2020 Oct;7(20): 2001800
    Wang C, Xu W, Chao Y, Liang M, Zhang F, Huang K.
      Chronic low-grade inflammation orchestrated by macrophages plays a critical role in metabolic chronic diseases, like obesity and atherosclerosis. However, the underlying mechanism remains to be elucidated. Here, the E3 ubiquitin ligase F-box/WD Repeat-Containing Protein 2 (FBXW2), the substrate-binding subunit of E3 ubiquitin ligase SCF (a complex of FBXW2, SKP1, and cullin-1), as an inflammatory mediator in macrophages, is identified. Myeloid-specific FBXW2 gene deficiency improves both obesity-associated with insulin resistance and atherosclerosis in murine models. The beneficial effects by FBXW2 knockout are accompanied by decreased proinflammatory responses and macrophage infiltration in the microenvironment. Mechanistically, it is identified that KH-type splicing regulatory protein (KSRP) is a new bona fide ubiquitin substrate of SCFFBXW2. Inhibition of KSRP prevents FBXW2-deficient macrophages from exerting a protective effect on inflammatory reactions, insulin resistance and plaque formation. Furthermore, it is demonstrated that the C-terminus (P3) of FBXW2 competitively ablates the function of FBXW2 in KSRP degradation and serves as an effective inhibitor of obesity and atherogenesis progression. Thus, the data strongly suggest that SCFFBXW2 is an important mediator in the context of metabolic diseases. The development of FBXW2 (P3)-mimicking inhibitors and small-molecular drugs specifically abrogating KSRP ubiquitination-dependent inflammatory responses are viable approaches for obesity and atherosclerosis treatment.
    Keywords:  FBXW2; KSRP; atherosclerosis; inflammation; insulin resistance; obesity
    DOI:  https://doi.org/10.1002/advs.202001800
  43. Genes Genomics. 2020 Oct 27.
    Yu G, Hyun S.
      As cells age, they lose their ability to properly fold proteins, maintain protein folding, and eliminate misfolded proteins, which leads to the accumulation of abnormal protein aggregates and loss of protein homeostasis (proteostasis). Loss of proteostasis can accelerate aging and the onset of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Mechanisms exist to prevent the detrimental effects of abnormal proteins that incorporate chaperones, autophagy, and the ubiquitin-proteasome system. These mechanisms are evolutionarily conserved across various species. Therefore, the effect of impaired proteostasis on aging has been studied using model organisms that are appropriate for aging studies. In this review, we focus on the relationship between proteostasis and aging, and factors that affect proteostasis in Drosophila. The manipulation of proteostasis can alter lifespan, modulate neurotoxicity, and delay the onset of neurodegeneration, indicating that proteostasis may be a novel pharmacological target for the development of treatments for various age-associated diseases.
    Keywords:  Aging; Autophagy; Chaperone; Drosophila; Proteasome; Proteostasis
    DOI:  https://doi.org/10.1007/s13258-020-01012-9
  44. Autophagy. 2020 Oct 28. 1-16
    Xu T, Nicolson S, Sandow JJ, Dayan S, Jiang X, Manning JA, Webb AI, Kumar S, Denton D.
      Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of our current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Here, using proteomic analysis of Drosophila larval midguts during degradation, we identify a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. We show that Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, we find that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. Our findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover.
    Keywords:   Drosophila ; Autophagy; cell death; lysosome; midgut; proteome
    DOI:  https://doi.org/10.1080/15548627.2020.1838105
  45. Nat Genet. 2020 Nov;52(11): 1208-1218
    Kinker GS, Greenwald AC, Tal R, Orlova Z, Cuoco MS, McFarland JM, Warren A, Rodman C, Roth JA, Bender SA, Kumar B, Rocco JW, Fernandes PACM, Mader CC, Keren-Shaul H, Plotnikov A, Barr H, Tsherniak A, Rozenblatt-Rosen O, Krizhanovsky V, Puram SV, Regev A, Tirosh I.
      Cultured cell lines are the workhorse of cancer research, but the extent to which they recapitulate the heterogeneity observed among malignant cells in tumors is unclear. Here we used multiplexed single-cell RNA-seq to profile 198 cancer cell lines from 22 cancer types. We identified 12 expression programs that are recurrently heterogeneous within multiple cancer cell lines. These programs are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-mesenchymal transition and protein metabolism. Most of these programs recapitulate those recently identified as heterogeneous within human tumors. We prioritized specific cell lines as models of cellular heterogeneity and used them to study subpopulations of senescence-related cells, demonstrating their dynamics, regulation and unique drug sensitivities, which were predictive of clinical response. Our work describes the landscape of heterogeneity within diverse cancer cell lines and identifies recurrent patterns of heterogeneity that are shared between tumors and specific cell lines.
    DOI:  https://doi.org/10.1038/s41588-020-00726-6
  46. FEBS Lett. 2020 Oct 27.
    Zhang W, Tao SS, Wang T, Zhang J, Liu X, Li YT, Chen H, Zhan YQ, Yu M, Ge CH, Li CY, Ren GM, Yang XM, Yin RH.
      BRCA1/BRCA2-containing complex subunit 3 (BRCC3) is a lysine 63-specific deubiquitinase involved in multiple biological processes, such as DNA repair and immune responses. However, the regulation mechanism for BRCC3 protein stability is still unknown. Here, we demonstrate that BRCC3 is mainly degraded through the ubiquitin-proteasome pathway. The HECT-type E3 ubiquitin ligase WWP2 modulates BRCC3 ubiquitination and degradation. ABRO1, a subunit of the BRCC36 isopeptidase complex (BRISC), competes with WWP2 to bind to BRCC3, thereby preventing WWP2-mediated BRCC3 ubiquitination and enhancing BRCC3 stability. Functionally, we show that lentivirus-mediated overexpression of WWP2 in murine macrophages inhibits NLRP3 inflammasome activation by decreasing BRCC3 protein level. This study provides the first insights into the regulation of BRCC3 stability and expands our knowledge about the physiological function of WWP2.
    Keywords:  ABRO1; BRCC3; NLRP3 inflammasome; WWP2; stability; ubiquitination
    DOI:  https://doi.org/10.1002/1873-3468.13970
  47. Cell Chem Biol. 2020 Oct 23. pii: S2451-9456(20)30384-6. [Epub ahead of print]
    Zavareh RB, Spangenberg SH, Woods A, Martínez-Peña F, Lairson LL.
      Cancer immunotherapies, including immune checkpoint blockade, have the potential to significantly impact treatments for diverse tumor types. At present, response failures and immune-related adverse events remain significant issues, which could be addressed using optimized combination therapies. Through a cell-based chemical screen of ∼200,000 compounds, we identified that HSP90 inhibitors robustly decrease PD-L1 surface expression, through a mechanism that appears to involve the regulation of master transcriptional regulators (i.e., STAT-3 and c-Myc). Interestingly, HSP90 inhibitors were found to also modulate the surface expression of additional checkpoint proteins (i.e., PD-L2). In the MC-38 syngeneic mouse tumor model, HSP90 inhibition was found to dramatically reduce PD-L1 surface expression on isolated live tumor cells and, consistent with recent findings, was found to increase the number of activated CD8+ T cells within the tumor microenvironment. These findings provide further rationale to explore HSP90 inhibitors as part of combination immunotherapies for the treatment of cancer.
    Keywords:  Programmed death-ligand 1; Programmed death-ligand 2; drug repurposing; flow cytometry-based screening; heat shock protein 90; immune checkpoint protein; immuno-oncology; phenotypic screening
    DOI:  https://doi.org/10.1016/j.chembiol.2020.10.005
  48. J Clin Invest. 2020 Oct 26. pii: 138234. [Epub ahead of print]
    Guo Y, Li L, Xu T, Guo X, Wang C, Li Y, Yang Y, Yang D, Sun B, Zhao X, Shao G, Qi X.
      The mechanism by which inflammasome activation is modulated remains unclear. In this study, we identified an AIM2-interacting protein, the E3 ubiquitin ligase HUWE1, which was also found to interact with NLRP3 and NLRC4 through the HIN domain of AIM2 and the NACHT domains of NLRP3 and NLRC4. The BH3 domain of HUWE1 was important for its interaction with NLRP3, AIM2, and NLRC4. Caspase-1 maturation, IL-1β release, and pyroptosis were reduced in Huwe1-deficient bone marrow-derived macrophages (BMDMs) compared with WT BMDMs in response to stimuli to induce NLRP3, NLRC4, and AIM2 inflammasome activation. Furthermore, the activation of NLRP3, NLRC4, and AIM2 inflammasomes in both mouse and human cells was remarkably reduced by treatment with the HUWE1 inhibitor BI8622. HUWE1 mediated the K27-linked polyubiquitination of AIM2, NLRP3, and NLRC4, which led to inflammasome assembly, ASC speck formation, and sustained caspase-1 activation. Huwe1-deficient mice had an increased bacterial burden and decreased caspase-1 activation and IL-1β production upon Salmonella, Francisella, or Acinetobacter baumannii infection. Our study provides insights into the mechanisms of inflammasome activation as well as a potential therapeutic target against bacterial infection.
    Keywords:  Immunology; Inflammation; Innate immunity; Macrophages; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI138234
  49. Elife. 2020 Oct 26. pii: e55865. [Epub ahead of print]9
    Pavlović N, Calitz C, Thanapirom K, Mazza G, Rombouts K, Gerwins P, Heindryckx F.
      Hepatocellular carcinoma (HCC) is a liver tumor that usually arises in patients with cirrhosis. Hepatic stellate cells are key players in the progression of HCC, as they create a fibrotic micro-environment and produce growth factors and cytokines that enhance tumor cell proliferation and migration. We assessed the role of endoplasmic reticulum (ER) stress in the cross-talk between stellate cells and HCC-cells. Mice with a fibrotic HCC were treated with the IRE1α-inhibitor 4μ8C, which reduced tumor burden and collagen deposition. By co-culturing HCC-cells with stellate cells, we found that HCC-cells activate IREα in stellate cells, thereby contributing to their activation. Inhibiting IRE1α blocked stellate cell activation, which then decreased proliferation and migration of tumor cells in different in vitro 2D and 3D co-cultures. In addition, we also observed cell-line specific direct effects of inhibiting IRE1α in tumor cells.
    Keywords:  cancer biology; mouse
    DOI:  https://doi.org/10.7554/eLife.55865
  50. FEBS Open Bio. 2020 Oct 27.
    János Engler M, Mimura J, Yamazaki S, Itoh K.
      Jun dimerization protein 2 (JDP2) is a bZip type transcription factor, which acts as a repressor or activator of several cellular processes, including cell differentiation and chromatin remodeling. Previously, we found that a stress-responsive transcription factor, known as activating transcription factor 4 (ATF4) enhances JDP2 gene expression in human astrocytoma U373MG and cervical cancer HeLa cells; however, the role of JDP2 in the ATF4-mediated stress response remained unclear. Here, we reported that siRNA-mediated JDP2 knockdown enhances the expression of several ATF4 target genes, including ASNS, and death receptors 4 and 5 (DR4 and DR5) in HeLa cells. In addition, the results of a transient reporter assay indicate that JDP2 overexpression represses ER stress-mediated DR5 promoter activation suggesting that JDP2 negatively regulates ATF4-mediated gene expression. Curiously, knockdown of JDP2 increases the sensitivity of cells to TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in cancer cells through DR4 and DR5. These results indicate that JDP2 functions as a negative feedback regulator of the ATF4 pathway and contributes to TRAIL resistance in cancer cells.
    Keywords:  ATF4; DR5; JDP2; TRAIL; cancer
    DOI:  https://doi.org/10.1002/2211-5463.13017
  51. Mol Oncol. 2020 Oct 25.
    Lankes K, Hassan ZZ, Doffo MJ, Schneeweis C, Lier S, Öllinger R, Rad R, Krämer OH, Keller U, Saur D, Reichert M, Schneider G, Wirth M.
      The myelocytomatosis oncogene (MYC) is an important driver in a subtype of pancreatic ductal adenocarcinoma (PDAC). However, MYC remains a challenging therapeutic target, therefore identifying druggable synthetic lethal interactions in MYC-active PDAC may lead to novel precise therapies. First, to identify networks with hyperactive MYC, we profiled transcriptomes of established human cell lines, murine primary PDAC cell lines and accessed publicly available repositories to analyze transcriptomes of primary human PDAC. Networks active in MYC hyperactive subtypes were analyzed by gene set enrichment analysis. Next, we performed an unbiased pharmacological screen to define MYC-associated vulnerabilities. Hits were validated by analysis of drug-response repositories and genetic gain- and loss-of-function experiments. In these experiments, we discovered that the proteasome inhibitor Bortezomib triggers a MYC-associated vulnerability. In addition, by integrating publicly available data, we found the unfolded protein response as a signature connected to MYC. Furthermore, increased sensitivity of MYC-hyperactive PDACs to Bortezomib was validated in genetically modified PDAC cells. In sum, we provide evidence that perturbing the ubiquitin proteasome system might be an option to target MYC hyperactive PDAC cells. Our data provide the rationale to further develop precise targeting of the ubiquitin-proteasome system as a subtype-specific therapeutic approach.
    Keywords:  MYC; UPR; UPS; apoptosis; pancreatic cancer; proteasome inhibitor
    DOI:  https://doi.org/10.1002/1878-0261.12835
  52. Science. 2020 Oct 29. pii: eabb5390. [Epub ahead of print]
    Esk C, Lindenhofer D, Haendeler S, Wester RA, Pflug F, Schroeder B, Bagley JA, Elling U, Zuber J, von Haeseler A, Knoblich JA.
      Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional (2D) cell culture hindering testing of gene functions requiring tissue context. Here we present CRISPR-LIneage tracing at Cellular resolution in Heterogenous Tissue (CRISPR-LICHT), enabling parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized Immediate Early Response 3 Interacting Protein 1 (IER3IP1) regulating the unfolded protein response (UPR) and extracellular matrix (ECM) protein secretion crucial for tissue integrity, with dysregulation resulting in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain size control.
    DOI:  https://doi.org/10.1126/science.abb5390
  53. Mol Biol Cell. 2020 Oct 28. mbcE20090591
    Casler JC, Zajac AL, Valbuena FM, Sparvoli D, Jeyifous O, Turkewitz AP, Horne-Badovinac S, Green WN, Glick BS.
      Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms. [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E20-09-0591
  54. Dev Cell. 2020 Oct 22. pii: S1534-5807(20)30795-4. [Epub ahead of print]
    Ardissone S, Kint N, Petrignani B, Panis G, Viollier PH.
      How cellular checkpoints couple the orderly assembly of macromolecular machines with cell-cycle progression is poorly understood. The alpha-proteobacterium Caulobacter crescentus assembles a single polar flagellum during each cell cycle. We discovered that the expression of multiple flagellin transcripts is licensed by a translational checkpoint responsive to a dual input signal: a secretion-competent hook-basal-body (HBB) structure and a surge in the FlaF secretion chaperone during cytokinesis, instructed by the cell-cycle program. We find that the unorthodox FljJ flagellin, one of the six flagellin paralogs, acts as a checkpoint linchpin, binding both FlaF and the FlbT translational regulator. FljJ recruits FlbT to inhibit translation at the 5' untranslated region in other flagellin transcripts before HBB assembly. Once FlaF is synthesized and stabilized, it directs FljJ secretion through the HBB, thereby separating FlbT from its co-activator and relieving translational inhibition. The FlbT/FlaF pair is wide spread and its functional properties are conserved in alpha-proteobacteria, including pathogens.
    Keywords:  Brucella melitensis; Caulobacter crescentus; FlaF; FlbT; FljJ; Tn-Seq; UTR; cell cycle; flagellum; morphological coupling; translational regulation
    DOI:  https://doi.org/10.1016/j.devcel.2020.10.005
  55. Mol Biol Cell. 2020 Oct 28. mbcE20080539
    Yang G, Banfield DK.
      Glycosylphosphatidylinositol-anchored proteins (GPI-APs) undergo extensive post-translational modifications and remodeling including the addition and subsequent removal of phospho-ethanolamine (EtNP) from mannose 1 (Man1) and mannose 2 (Man2) of the glycan moiety. Removal of EtNP from Man1 is catalyzed by Cdc1p, an event that has previously been considered to occur in the ER. We establish that Cdc1p is in fact a cis / medial Golgi membrane protein that relies on COPI coatomer for its retention in this organelle. We also determine that Cdc1p does not cycle between the Golgi and the ER, and consistent with this finding, when expressed at endogenous levels ER-localized Cdc1p-HDEL is unable to support the growth of cdc1Δ cells. Our cdc1 temperature-sensitive alleles are defective in the transport of a prototypical GPI-AP - Gas1p to the cell surface, a finding we posit reveals a novel Golgi-localized quality control warrant. Thus, yeast cells scrutinize GPI-APs in the ER and also in the Golgi, where removal of EtNP from Man2 (via Ted1p in the ER) and from Man1 (by Cdc1p in the Golgi) functions as a quality assurance signal.
    DOI:  https://doi.org/10.1091/mbc.E20-08-0539
  56. Autophagy. 2020 Oct 28. 1-16
    Watzlawik JO, Hou X, Truban D, Ramnarine C, Barodia SK, Gendron TF, Heckman MG, DeTure M, Siuda J, Wszolek ZK, Scherzer CR, Ross OA, Bu G, Dickson DW, Goldberg MS, Fiesel FC, Springer W.
      Mitochondrial dysfunction is an early, imminent event in neurodegenerative disorders including Parkinson disease (PD) and Alzheimer disease (AD). The enzymatic pair PINK1 and PRKN/Parkin recognize and transiently label damaged mitochondria with ubiquitin (Ub) phosphorylated at Ser65 (p-S65-Ub) as a signal for degradation via the autophagy-lysosome system (mitophagy). Despite its discovery in cell culture several years ago, robust and quantitative detection of altered mitophagy in vivo has remained challenging. Here we developed a sandwich ELISA targeting p-S65-Ub with the goal to assess mitophagy levels in mouse brain and in human clinical and pathological samples. We characterized five total Ub and four p-S65-Ub antibodies by several techniques and found significant differences in their ability to recognize phosphorylated Ub. The most sensitive antibody pair detected recombinant p-S65-Ub chains in the femtomolar to low picomolar range depending on the poly-Ub chain linkage. Importantly, this ELISA was able to assess very low baseline mitophagy levels in unstressed human cells and in brains from wild-type and prkn knockout mice as well as elevated p-S65-Ub levels in autopsied frontal cortex from AD patients vs. control cases. Moreover, the assay allowed detection of p-S65-Ub in blood plasma and was able to discriminate between PINK1 mutation carriers and controls. In summary, we developed a robust and sensitive tool to measure mitophagy levels in cells, tissue, and body fluids. Our data strongly support the idea that the stress-activated PINK1-PRKN mitophagy pathway is constitutively active in mice and humans under unstimulated, physiological and elevated in diseased, pathological conditions. Abbreviations: Ab: antibody; AD: Alzheimer disease; AP: alkaline phosphatase; CV: coefficient of variation; ECL: electrochemiluminescence; KO: knockout; LoB: Limit of Blank; LoD: Limit of Detection; LoQ: Limit of Quantification; MSD: meso scale discovery; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated ubiquitin at serine 65; Std.Dev.: standard deviation; Ub: ubiquitin; WT: wild type.
    Keywords:  Alzheimer disease; PINK1; PRKN; Parkin; Parkinson disease; autophagy; mitophagy; ubiquitin
    DOI:  https://doi.org/10.1080/15548627.2020.1834712
  57. Int J Biol Sci. 2020 ;16(14): 2727-2740
    Zhang Y, Qian H, Wu B, You S, Wu S, Lu S, Wang P, Cao L, Zhang N, Sun Y.
      Protein ubiquitination represents a critical modification occurring after translation. E3 ligase catalyzes the covalent binding of ubiquitin to the protein substrate, which could be degraded. Ubiquitination as an important protein post-translational modification is closely related to cardiovascular disease. The NEDD4 family, belonging to HECT class of E3 ubiquitin ligases can recognize different substrate proteins, including PTEN, ENaC, Nav1.5, SMAD2, PARP1, Septin4, ALK1, SERCA2a, TGFβR3 and so on, via the WW domain to catalyze ubiquitination, thus participating in multiple cardiovascular-related disease such as hypertension, arrhythmia, myocardial infarction, heart failure, cardiotoxicity, cardiac hypertrophy, myocardial fibrosis, cardiac remodeling, atherosclerosis, pulmonary hypertension and heart valve disease. However, there is currently no review comprehensively clarifying the important role of NEDD4 family proteins in the cardiovascular system. Therefore, the present review summarized recent studies about NEDD4 family members in cardiovascular disease, providing novel insights into the prevention and treatment of cardiovascular disease. In addition, assessing transgenic animals and performing gene silencing would further identify the ubiquitination targets of NEDD4. NEDD4 quantification in clinical samples would also constitute an important method for determining NEDD4 significance in cardiovascular disease.
    Keywords:  NEDD4 E3 ligases; cardiovascular disease; post translation modification; ubiquitin proteasome system
    DOI:  https://doi.org/10.7150/ijbs.48437
  58. Mol Cancer Res. 2020 Oct 26. pii: molcanres.0480.2019. [Epub ahead of print]
    Nath A, Oak A, Chen KY, Li I, Splichal RC, Portis J, Foster S, Walton SP, Chan C.
      Elevated uptake of saturated fatty acid palmitate is associated with metastatic progression of cancer cells; however, the precise signaling mechanism behind the phenomenon is unclear. The loss of cell adhesion proteins, such as desmoplakin (DSP), is a key driving event in the transformation of cancer cells to more aggressive phenotypes. Here we investigated the mechanism by which palmitate induces the loss of DSP in liver and breast cancer cells. We propose that palmitate activates the IRE1-XBP1 branch of the endoplasmic reticulum (ER) stress pathway to upregulate the ZEB transcription factor, leading to transcriptional repression of DSP. Using liver and breast cancer cells treated with palmitate, we found loss of DSP leads to increased cell migration independent of E-cadherin. We report that the ZEB family of transcription factors function as direct transcriptional repressors of DSP. CRISPR-mediated knockdown of IRE1 confirmed that the transcription of ZEB, loss of DSP, and enhanced migration in the presence of palmitate is dependent on the IRE1-XBP1 pathway. Additionally, by analyzing the somatic expression and copy number variation profiles of over 11,000 tumor samples, we corroborate our hypothesis and establish the clinical relevance of DSP loss via ZEB in human cancers. Implications: Provides mechanistic link on palmitate-induced activation of IRE1α to cancer cell migration.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-19-0480