bims-proteo Biomed News
on Proteostasis
Issue of 2020‒05‒03
38 papers selected by
Eric Chevet
INSERM
  1. Characterization of Endoplasmic Reticulum (ER) in Human Pluripotent Stem Cells Revealed Increased Susceptibility to Cell Death upon ER Stress.
  2. Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly.
  3. Ezetimibe Prevents Ischemia/Reperfusion-Induced Oxidative Stress and Up-Regulates Nrf2/ARE and UPR Signaling Pathways.
  4. Bacterial rhomboid proteases mediate quality control of orphan membrane proteins.
  5. Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress.
  6. Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis.
  7. Endoplasmic reticulum stress mediates resistance to BCL-2 inhibitor in uveal melanoma cells.
  8. Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response.
  9. The Deubiquitinating Enzyme USP20 Regulates the TNFα-Induced NF-κB Signaling Pathway through Stabilization of p62.
  10. Characterization of a C-Terminal SUMO-Interacting Motif Present in Select PIAS-Family Proteins.
  11. Regulating the regulators: role of phosphorylation in modulating the function of the GBF1/BIG family of Sec7 ARF-GEFs.
  12. Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes.
  13. Correction of Niemann-Pick type C1 trafficking and activity with the histone deacetylase inhibitor valproic acid.
  14. Ligand-dependent downregulation of MR1 cell surface expression.
  15. Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy.
  16. Torsin ATPase deficiency leads to defects in nuclear pore biogenesis and sequestration of MLF2.
  17. Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing.
  18. Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation.
  19. Hop depletion reduces HSF1 levels and activity and coincides with reduced stress resilience.
  20. BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates.
  21. The DNA-binding activity of USP1-associated factor 1 is required for efficient RAD51-mediated homologous DNA pairing and homology-directed DNA repair.
  22. Sec22b determines Weibel-Palade body length by controlling anterograde ER-Golgi transport.
  23. Facultative protein selenation regulates redox sensitivity, adipose tissue thermogenesis, and obesity.
  24. The structural basis for inhibition of ribosomal translocation by viomycin.
  25. UHRF1 Controls Thymocyte Fate Decisions through the Epigenetic Regulation of EGR1 Expression.
  26. Regulation of the Golgi apparatus via GOLPH3-mediated new selective autophagy.
  27. ER Stress Regulates Immunosuppressive Function of Myeloid Derived Suppressor Cells in Leprosy that Can Be Overcome in the Presence of IFN-γ.
  28. Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly.
  29. Behind closed gates - chaperones and charged residues determine protein fate.
  30. Hyd ubiquitinates the NF-κB co-factor Akirin to operate an effective immune response in Drosophila.
  31. Synthetic post-translational modifications of elongation factor P using the ligase EpmA.
  32. Derlins with scissors: primordial ERAD in bacteria.
  33. Cullin-RING Ligase 5: Functional characterization and its role in human cancers.
  34. Spliced X-box binding protein 1 induces liver cancer cell death via activating the Mst1-JNK-mROS signalling pathway.
  35. Cellular mechanisms and treatments for chemobrain: insight from aging and neurodegenerative diseases.
  36. N-glycosylation controls inflammatory licensing-triggered PD-L1 upregulation in human mesenchymal stromal cells.
  37. A modified lysosomal organelle mediates nonlytic egress of reovirus.
  38. Caspase-1 cleaves Bid to release mitochondrial SMAC and drive secondary necrosis in the absence of GSDMD.
  1. Cells. 2020 Apr 26. pii: E1078. [Epub ahead of print]9(5):
    Characterization of Endoplasmic Reticulum (ER) in Human Pluripotent Stem Cells Revealed Increased Susceptibility to Cell Death upon ER Stress.
    Tae Won Ha, Ji Hun Jeong, HyeonSeok Shin, Hyun Kyu Kim, Jeong Suk Im, Byung Hoo Song, Jacob Hanna, Jae Sang Oh, Dong-Hun Woo, Jaeseok Han, Man Ryul Lee.
      Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a well-orchestrated program for differentiation and self-renewal. However, the structural features of unique proteostatic-maintaining mechanisms in hPSCs and their features, distinct from those of differentiated cells, in response to cellular stress remain unclear. We evaluated and compared the morphological features and stress response of hPSCs and fibroblasts. Compared to fibroblasts, electron microscopy showed simpler/fewer structures with fewer networks in the endoplasmic reticulum (ER) of hPSCs, as well as lower expression of ER-related genes according to meta-analysis. As hPSCs contain low levels of binding immunoglobulin protein (BiP), an ER chaperone, thapsigargin treatment sharply increased the gene expression of the unfolded protein response. Thus, hPSCs with decreased chaperone function reacted sensitively to ER stress and entered apoptosis faster than fibroblasts. Such ER stress-induced apoptotic processes were abolished by tauroursodeoxycholic acid, an ER-stress reliever. Hence, our results revealed that as PSCs have an underdeveloped structure and express fewer BiP chaperone proteins than somatic cells, they are more susceptible to ER stress-induced apoptosis in response to stress.
    Keywords:  C/EBP homologous protein (CHOP); ER stress; binding immunoglobulin protein (BiP); endoplasmic reticulum (ER); human pluripotent stem cells; proteostasis
    DOI:  https://doi.org/10.3390/cells9051078
  2. J Biol Chem. 2020 May 01. pii: jbc.RA120.012964. [Epub ahead of print]
    Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly.
    David B Melville, Sean Studer, Randy Schekman.
      Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum (ER) to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the ER membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here, we identified two amino acid clusters that have conserved, SAR1 paralog-specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.
    Keywords:  COPII; GTPase; SAR1A; SAR1B; SEC23; SEC31; apolipoprotein; intracellular trafficking; membrane trafficking; secretion
    DOI:  https://doi.org/10.1074/jbc.RA120.012964
  3. Antioxidants (Basel). 2020 Apr 23. pii: E349. [Epub ahead of print]9(4):
    Ezetimibe Prevents Ischemia/Reperfusion-Induced Oxidative Stress and Up-Regulates Nrf2/ARE and UPR Signaling Pathways.
    Denise Peserico, Chiara Stranieri, Ulisse Garbin, Chiara Mozzini C, Elisa Danese, Luciano Cominacini, Anna M Fratta Pasini.
      BACKGROUND: While reperfusion is crucial for survival after an episode of ischemia, it also causes oxidative stress. Nuclear factor-E2-related factor 2 (Nrf2) and unfolded protein response (UPR) are protective against oxidative stress and endoplasmic reticulum (ER) stress. Ezetimibe, a cholesterol absorption inhibitor, has been shown to activate the AMP-activated protein kinase (AMPK)/Nrf2 pathway. In this study we evaluated whether Ezetimibe affects oxidative stress and Nrf2 and UPR gene expression in cellular models of ischemia-reperfusion (IR).METHODS: Cultured cells were subjected to simulated IR with or without Ezetimibe.
    RESULTS: IR significantly increased reactive oxygen species (ROS) production and the percentage of apoptotic cells without the up-regulation of Nrf2, of the related antioxidant response element (ARE) gene expression or of the pro-survival UPR activating transcription factor 6 (ATF6) gene, whereas it significantly increased the pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP). Ezetimibe significantly decreased the cellular ROS formation and apoptosis induced by IR. These effects were paralleled by the up-regulation of Nrf2/ARE and ATF6 gene expression and by a down-regulation of CHOP. We also found that Nrf2 activation was dependent on AMPK, since Compound C, a pan inhibitor of p-AMPK, blunted the activation of Nrf2.
    CONCLUSIONS: Ezetimibe counteracts IR-induced oxidative stress and induces Nrf2 and UPR pathway activation.
    Keywords:  ER stress; Ezetimibe; Nrf2; ischemia-reperfusion; oxidative stress
    DOI:  https://doi.org/10.3390/antiox9040349
  4. EMBO J. 2020 Apr 27. e102922
    Bacterial rhomboid proteases mediate quality control of orphan membrane proteins.
    Guangyu Liu, Stephen E Beaton, Adam G Grieve, Rhiannon Evans, Miranda Rogers, Kvido Strisovsky, Fraser A Armstrong, Matthew Freeman, Rachel M Exley, Christoph M Tang.
      Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic rhomboids has remained enigmatic. Here, we show that the Shigella sonnei rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.
    Keywords:   Shigella ; intramembrane proteolysis; membrane protein complexes; quality control; rhomboid
    DOI:  https://doi.org/10.15252/embj.2019102922
  5. J Cell Biol. 2020 Jul 06. pii: e201909165. [Epub ahead of print]219(7):
    Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress.
    Nurulain Ho, Wei Sheng Yap, Jiaming Xu, Haoxi Wu, Jhee Hong Koh, Wilson Wen Bin Goh, Bhawana George, Shu Chen Chong, Stefan Taubert, Guillaume Thibault.
      Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated, and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention.
    DOI:  https://doi.org/10.1083/jcb.201909165
  6. J Cell Biol. 2020 Jul 06. pii: e201910177. [Epub ahead of print]219(7):
    Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis.
    Vineet Choudhary, Ola El Atab, Giulia Mizzon, William A Prinz, Roger Schneiter.
      Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER-LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG-synthases and additional LD biogenesis factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.
    DOI:  https://doi.org/10.1083/jcb.201910177
  7. Cell Death Discov. 2020 ;6 22
    Endoplasmic reticulum stress mediates resistance to BCL-2 inhibitor in uveal melanoma cells.
    Lara Bellini, Thomas Strub, Nadia Habel, Charlotte Pandiani, Sandrine Marchetti, Arnaud Martel, Stéphanie Baillif, Béatrice Bailly-Maitre, Philippe Gual, Robert Ballotti, Corine Bertolotto.
      To address unmet clinical need for uveal melanomas, we assessed the effects of BH3-mimetic molecules, the ABT family, known to exert pro-apoptotic activities in cancer cells. Our results uncovered that ABT-263 (Navitoclax), a potent and orally bioavailable BCL-2 family inhibitor, induced antiproliferative effects in metastatic human uveal melanoma cells through cell cycle arrest at the G0/G1 phase, loss of mitochondrial membrane potential, and subsequently apoptotic cell death monitored by caspase activation and poly-ADP ribose polymerase cleavage. ABT-263-mediated reduction in tumor growth was also observed in vivo. We observed in some cells that ABT-263 treatment mounted a pro-survival response through activation of the ER stress signaling pathway. Blocking the PERK signaling pathway increased the pro-apoptotic ABT-263 effect. We thus uncovered a resistance mechanism in uveal melanoma cells mediated by activation of endoplasmic reticulum stress pathway. Therefore, our study identifies ABT-263 as a valid therapeutic option for patients suffering from uveal melanoma.
    Keywords:  Cell death; Eye cancer
    DOI:  https://doi.org/10.1038/s41420-020-0259-2
  8. J Mol Cell Cardiol. 2020 Apr 24. pii: S0022-2828(20)30094-8. [Epub ahead of print]
    Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response.
    Erik A Blackwood, Donna J Thuerauf, Miroslava Stastna, Haley Stephens, Zoe Sand, Amber Pentoney, Khalid Azizi, Tobias Jakobi, Jennifer E Van Eyk, Hugo A Katus, Christopher C Glembotski, Shirin Doroudgar.
      The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases SR/ER calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.
    Keywords:  Cardiac myocyte death; Cardiokine; Cardioprotection; ER stress; Heart failure; Proteostasis
    DOI:  https://doi.org/10.1016/j.yjmcc.2020.04.012
  9. Int J Mol Sci. 2020 Apr 28. pii: E3116. [Epub ahead of print]21(9):
    The Deubiquitinating Enzyme USP20 Regulates the TNFα-Induced NF-κB Signaling Pathway through Stabilization of p62.
    Jihoon Ha, Minbeom Kim, Dongyeob Seo, Jin Seok Park, Jaewon Lee, Jinjoo Lee, Seok Hee Park.
      p62/sequestosome-1 is a scaffolding protein involved in diverse cellular processes such as autophagy, oxidative stress, cell survival and death. It has been identified to interact with atypical protein kinase Cs (aPKCs), linking these kinases to NF-κB activation by tumor necrosis factor α (TNFα). The diverse functions of p62 are regulated through post-translational modifications of several domains within p62. Among the enzymes that mediate these post-translational modifications, little is known about the deubiquitinating enzymes (DUBs) that remove ubiquitin chains from p62, compared to the E3 ligases involved in p62 ubiquitination. In this study, we first demonstrate a role of ubiquitin-specific protease USP20 in regulating p62 stability in TNFα-mediated NF-κB activation. USP20 specifically binds to p62 and acts as a positive regulator for NF-κB activation by TNFα through deubiquitinating lysine 48 (K48)-linked polyubiquitination, eventually contributing to cell survival. Furthermore, depletion of USP20 disrupts formation of the atypical PKCζ-RIPK1-p62 complex required for TNFα-mediated NF-κB activation and significantly increases the apoptosis induced by TNFα plus cycloheximide or TNFα plus TAK1 inhibitor. These findings strongly suggest that the USP20-p62 axis plays an essential role in NF-κB-mediated cell survival induced by the TNFα-atypical PKCζ signaling pathway.
    Keywords:  apoptosis; cell survival; nuclear factor-κB; p62; tumor necrosis factor α; ubiquitin-specific protease 20
    DOI:  https://doi.org/10.3390/ijms21093116
  10. Structure. 2020 Apr 22. pii: S0969-2126(20)30121-0. [Epub ahead of print]
    Characterization of a C-Terminal SUMO-Interacting Motif Present in Select PIAS-Family Proteins.
    Mathieu Lussier-Price, Xavier H Mascle, Laurent Cappadocia, Rui Kamada, Kazuyasu Sakaguchi, Haytham M Wahba, James G Omichinski.
      The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how post-translational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.
    Keywords:  BRET; ITC; Mre11; PIAS-family proteins; SUMO; SUMO-interacting motif; Siz2; acetylation; crystallography; phosphorylation
    DOI:  https://doi.org/10.1016/j.str.2020.04.002
  11. FEBS Lett. 2020 Apr 25.
    Regulating the regulators: role of phosphorylation in modulating the function of the GBF1/BIG family of Sec7 ARF-GEFs.
    Kendall Walton, Andre Leier, Elizabeth Sztul.
      Membrane traffic between secretory and endosomal compartments is vesicle-mediated and must be tightly balanced to maintain a physiological compartment size. Vesicle formation is initiated by guanine nucleotide exchange factors (GEFs) that activate the ARF family of small GTPases. Regulatory mechanisms, including reversible phosphorylation, allow ARF-GEFs to support vesicle formation only at the right time and place in response to cellular needs. Here, we review current knowledge of how the GBF1/BIG family of ARF-GEFs is influenced by phosphorylation and use predictive paradigms to propose new regulatory paradigms. We describe a conserved cluster of phosphorylation sites within the N-terminal domains of the GBF1/BIG ARF-GEFs and suggest that these sites may respond to homeostatic signals related to cell growth and division. In the C-terminal region, GBF1 shows phosphorylation sites clustered differently as compared with the similar configuration found in both BIG1 and BIG2. Despite this similarity, BIG1 and BIG2 phosphorylation patterns are divergent in other domains. The different clustering of phosphorylation sites suggests that the non-conserved sites may represent distinct regulatory nodes and specify the function of GBF1, BIG1, and BIG2.
    Keywords:  ARF; BIG1; BIG2; GBF1; GEF; Sec7; membrane traffic; phosphorylation
    DOI:  https://doi.org/10.1002/1873-3468.13798
  12. Life Sci Alliance. 2020 Jun;pii: e202000700. [Epub ahead of print]3(6):
    Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes.
    Huda Jumaa, Marieta Caganova, Ellen J McAllister, Laura Hoenig, Xiaocui He, Deniz Saltukoglu, Kathrin Brenker, Markus Köhler, Ruth Leben, Anja E Hauser, Raluca Niesner, Klaus Rajewsky, Michael Reth, Julia Jellusova.
      The major function of B lymphocytes is to sense antigens and to produce protective antibodies after activation. This function requires the expression of a B-cell antigen receptor (BCR), and evolutionary conserved mechanisms seem to exist that ensure that B cells without a BCR do not develop nor survive in the periphery. Here, we show that the loss of BCR expression on Burkitt lymphoma cells leads to decreased mitochondrial function and impaired metabolic flexibility. Strikingly, this phenotype does not result from the absence of a classical Syk-dependent BCR signal but rather from compromised ER expansion. We show that the reexpression of immunoglobulins (Ig) in the absence of the BCR signaling subunits Igα and Igβ rescues the observed metabolic defects. We demonstrate that immunoglobulin expression is needed to maintain ER homeostasis not only in lymphoma cells but also in resting B cells. Our study provides evidence that the expression of BCR components, which is sensed in the ER and shapes mitochondrial function, represents a novel mechanism of metabolic control in B cells.
    DOI:  https://doi.org/10.26508/lsa.202000700
  13. J Biol Chem. 2020 Apr 30. pii: jbc.RA119.010524. [Epub ahead of print]
    Correction of Niemann-Pick type C1 trafficking and activity with the histone deacetylase inhibitor valproic acid.
    Kanagaraj Subramanian, Darren M Hutt, Samantha M Scott, Vijay Gupta, Shu Mao, William E Balch.
      Niemann-Pick type C (NPC) disease is primarily caused by mutations in the NPC1 gene and is characterized by the accumulation of unesterified cholesterol and lipids in the late endosomal (LE) and lysosomal (Ly) compartments. The most prevalent disease-linked mutation is the I1061T variant of NPC1, which exhibits defective folding and trafficking from the endoplasmic reticulum to the LE/Ly compartments. We now show that the FDA-approved histone deacetylase inhibitor (HDACi) valproic acid (VPA) corrects the folding and trafficking defect associated with I1061T-NPC1 leading to restoration of cholesterol homeostasis, an effect that is largely driven by a reduction in HDAC7 expression. The VPA-mediated trafficking correction is in part associated with an increase in the acetylation of lysine residues in the cysteine-rich domain of NPC1. The HDACi-mediated correction is synergistically improved by combining it with the FDA-approved anti-malarial, chloroquine, a known lysosomotropic compound, which improved the stability of the LE/Ly-localized fraction of the I1061T variant. We posit that combining the activity of VPA, to modulate epigenetically the  cellular acetylome, with chloroquine, to alter the lysosomal environment to favor stability of the trafficked I1061T variant protein, can have a significant therapeutic benefit in patients carrying at least one copy of the I1061T variant of NPC1, the most common disease-associated mutation leading to NPC disease. Given its ability to cross the blood brain barrier, we posit VPA provides a potential mechanism to improve the response to 2-hydroxypropyl-b-cyclodextrin, by restoring a functional NPC1 to the cholesterol managing compartment as an adjunct therapy.
    Keywords:  Cholesterol homeostasis; Choloroquine; NPC1; Niemann-Pick type C disease; Valproic acid; cholesterol; histone deacetylase inhibitor (HDAC inhibitor) (HDI); intracellular trafficking; lysosome; neurodegenerative disease
    DOI:  https://doi.org/10.1074/jbc.RA119.010524
  14. Proc Natl Acad Sci U S A. 2020 Apr 27. pii: 202003136. [Epub ahead of print]
    Ligand-dependent downregulation of MR1 cell surface expression.
    Mariolina Salio, Wael Awad, Natacha Veerapen, Claudia Gonzalez-Lopez, Corinna Kulicke, Dominic Waithe, Anne W J Martens, David M Lewinsohn, Judith V Hobrath, Liam R Cox, Jamie Rossjohn, Gurdyal S Besra, Vincenzo Cerundolo.
      The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A'-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.
    DOI:  https://doi.org/10.1073/pnas.2003136117
  15. EMBO Rep. 2020 Apr 26. e48192
    Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy.
    Andrea Martello, Angela Lauriola, David Mellis, Elisa Parish, John C Dawson, Lisa Imrie, Martina Vidmar, Noor Gammoh, Tijana Mitić, Mairi Brittan, Nicholas L Mills, Neil O Carragher, Domenico D'Arca, Andrea Caporali.
      Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome-dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP-depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF-κB inhibition. Moreover, Tchp knock-out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy-mediated mechanism.
    Keywords:   GABARAP ; SQSTM1/p62; autophagy; centriolar satellites; endothelial cells
    DOI:  https://doi.org/10.15252/embr.201948192
  16. J Cell Biol. 2020 Jun 01. pii: e201910185. [Epub ahead of print]219(6):
    Torsin ATPase deficiency leads to defects in nuclear pore biogenesis and sequestration of MLF2.
    Anthony J Rampello, Ethan Laudermilch, Nidhi Vishnoi, Sarah M Prophet, Lin Shao, Chenguang Zhao, C Patrick Lusk, Christian Schlieker.
      Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify myeloid leukemia factor 2 (MLF2) as a luminal component of the bleb. Using an MLF2-based live-cell imaging platform, we demonstrate that nuclear envelope blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG-nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin-deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents aberrant NPC intermediates.
    DOI:  https://doi.org/10.1083/jcb.201910185
  17. Cell Death Differ. 2020 Apr 27.
    Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing.
    Björn Friedhelm Vahsen, Vinicius Toledo Ribas, Jonas Sundermeyer, Alexander Boecker, Vivian Dambeck, Christof Lenz, Orr Shomroni, Lucas Caldi Gomes, Lars Tatenhorst, Elisabeth Barski, Anna-Elisa Roser, Uwe Michel, Henning Urlaub, Gabriela Salinas, Mathias Bähr, Jan Christoph Koch, Paul Lingor.
      Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.
    DOI:  https://doi.org/10.1038/s41418-020-0543-y
  18. Proc Natl Acad Sci U S A. 2020 Apr 30. pii: 201918459. [Epub ahead of print]
    Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation.
    Shanta Karki, Kathrina Castillo, Zhaolan Ding, Olivia Kerr, Teresa M Lamb, Cheng Wu, Matthew S Sachs, Deborah Bell-Pedersen.
      The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.
    Keywords:  Neurospora crassa; circadian clock; cpc-3; eIF2α; translation initiation
    DOI:  https://doi.org/10.1073/pnas.1918459117
  19. Biochem Biophys Res Commun. 2020 Apr 22. pii: S0006-291X(20)30798-1. [Epub ahead of print]
    Hop depletion reduces HSF1 levels and activity and coincides with reduced stress resilience.
    Abantika Chakraborty, Adrienne Lesley Edkins.
      Heat-shock factor 1 (HSF1) regulates the transcriptional response to stress and controls expression of molecular chaperones required for cell survival. Here we report that HSF1 is regulated by the abundance of the Hsp70-Hsp90 organizing protein (Hop/STIP1). HSF1 levels were significantly reduced in Hop-depleted HEK293T cells. HSF1 transcriptional activity at the Hsp70 promoter, and binding of a biotinylated HSE oligonucleotide under both basal and heat-shock conditions were significantly reduced. Hop-depleted HEK293T cells were more sensitive to the HSF1 inhibitor KRIBB11 and showed reduced short-term proliferation, and reduced long-term survival under basal and heat-shock conditions. HSF1 nuclear localization was reduced in response to heat-shock and the nuclear staining pattern in Hop-depleted cells was punctate. Taken together, these data suggest that Hop regulates HSF1 function under both basal and stress conditions through a mechanism involving changes in levels, activity and subcellular localization, and coincides with reduced cellular fitness.
    Keywords:  HSF1; Heat-shock; Hop; KRIBB11; STIP1
    DOI:  https://doi.org/10.1016/j.bbrc.2020.04.072
  20. Mol Cell. 2020 Apr 14. pii: S1097-2765(20)30227-6. [Epub ahead of print]
    BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates.
    Nadra Al-Husini, Dylan T Tomares, Zechariah J Pfaffenberger, Nisansala S Muthunayake, Mohammad A Samad, Tiancheng Zuo, Obaidah Bitar, James R Aretakis, Mohammed-Husain M Bharmal, Alisa Gega, Julie S Biteen, W Seth Childers, Jared M Schrader.
      Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.
    Keywords:  BR-bodies; RNP granules; Ribonuclease E; biomolecular condensates; mRNA decay; phase separation; ribonucleoprotein
    DOI:  https://doi.org/10.1016/j.molcel.2020.04.001
  21. J Biol Chem. 2020 Apr 29. pii: jbc.RA120.013714. [Epub ahead of print]
    The DNA-binding activity of USP1-associated factor 1 is required for efficient RAD51-mediated homologous DNA pairing and homology-directed DNA repair.
    Fengshan Liang, Adam S Miller, Caroline Tang, David Maranon, Elizabeth A Williamson, Robert Hromas, Claudia Wiese, Weixing Zhao, Patrick Sung, Gary M Kupfer.
      USP1-associated factor 1 (UAF1) is an integral component of the RAD51-associated protein 1 (RAD51AP1)-UAF1-ubiquitin-specific peptidase 1 (USP1) trimeric deubiquitinase complex. This complex acts on DNA bound, monoubiquitinated FA complementation group D2 (FANCD2) protein in the Fanconi anemia pathway of the DNA damage response. Moreover, RAD51AP1 and UAF1 cooperate to enhance homologous DNA pairing mediated by the recombinase RAD51 in DNA repair via the homologous recombination (HR) pathway. However, whereas the DNA-binding activity of RAD51AP1 has been shown to be important for RAD51-mediated homologous DNA pairing and HR-mediated DNA repair, the role of DNA binding by UAF1 in these processes is unclear. We have isolated mutant UAF1 variants that are impaired in DNA binding and tested them together with RAD51AP1 in RAD51-mediated HR. This biochemical analysis revealed that the DNA-binding activity of UAF1 is indispensable for enhanced RAD51 recombinase activity within the context of the UAF1-RAD51AP1 complex. In cells, DNA-binding deficiency of UAF1 increased DNA damage sensitivity and impaired HR efficiency, suggesting that UAF1 and RAD51AP1 have coordinated roles in DNA binding during HR and DNA damage repair. Our findings show that, even though UAF1's DNA-binding activity is redundant with that of RAD51AP1 in FANCD2 deubiquitination, it is required for efficient HR-mediated chromosome damage repair.
    Keywords:  DNA binding protein; DNA damage; DNA damage response; DNA repair; Fanconi anemia; UAF1-RAD51AP1; deubiquitination; homologous recombination; interstrand cross-linking (ICL); ubiquitin-specific peptidase 1 (USP1)
    DOI:  https://doi.org/10.1074/jbc.RA120.013714
  22. Haematologica. 2020 Mar 26. pii: haematol.2019.242727. [Epub ahead of print]
    Sec22b determines Weibel-Palade body length by controlling anterograde ER-Golgi transport.
    Ellie Karampini, Petra E Bürgisser, Jenny Olins, Aat A Mulder, Carolina R Jost, Dirk Geerts, Jan Voorberg, Ruben Bierings.
      Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles, so-called Weibel-Palade bodies (WPBs). Hemostatic activity of VWF is strongly tied to WPB length, but how endothelial cells control the dimensions of their WPBs is unclear. In this study we used a targeted shRNA screen to identify the longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology along with disintegration of the Golgi and dilation of rough ER (rER) cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rER and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPBs, and thus adhesive activity of its cargo VWF, is determined by the rate of anterograde transport between ER and Golgi, which depends on Sec22b-containing SNARE complexes.
    Keywords:  ER/Golgi trafficking; SNARE proteins; VWF trafficking; Vascular Wall Biology and Platelet Adhesion; WPB biogenesis
    DOI:  https://doi.org/10.3324/haematol.2019.242727
  23. Proc Natl Acad Sci U S A. 2020 May 01. pii: 202001387. [Epub ahead of print]
    Facultative protein selenation regulates redox sensitivity, adipose tissue thermogenesis, and obesity.
    Mark P Jedrychowski, Gina Z Lu, John Szpyt, Marco Mariotti, Ryan Garrity, Joao A Paulo, Devin K Schweppe, Dina Laznik-Bogoslavski, Lawrence Kazak, Michael P Murphy, Vadim N Gladyshev, Steven P Gygi, Edward T Chouchani, Bruce M Spiegelman.
      Oxidation of cysteine thiols by physiological reactive oxygen species (ROS) initiates thermogenesis in brown and beige adipose tissues. Cellular selenocysteines, where sulfur is replaced with selenium, exhibit enhanced reactivity with ROS. Despite their critical roles in physiology, methods for broad and direct detection of proteogenic selenocysteines are limited. Here we developed a mass spectrometric method to interrogate incorporation of selenium into proteins. Unexpectedly, this approach revealed facultative incorporation of selenium as selenocysteine or selenomethionine into proteins that lack canonical encoding for selenocysteine. Selenium was selectively incorporated into regulatory sites on key metabolic proteins, including as selenocysteine-replacing cysteine at position 253 in uncoupling protein 1 (UCP1). This facultative utilization of selenium was initiated by increasing cellular levels of organic, but not inorganic, forms of selenium. Remarkably, dietary selenium supplementation elevated facultative incorporation into UCP1, elevated energy expenditure through thermogenic adipose tissue, and protected against obesity. Together, these findings reveal the existence of facultative protein selenation, which correlates with impacts on thermogenic adipocyte function and presumably other biological processes as well.
    Keywords:  ROS; brown adipose tissue; cysteine; selenocysteine
    DOI:  https://doi.org/10.1073/pnas.2001387117
  24. Proc Natl Acad Sci U S A. 2020 Apr 27. pii: 202002888. [Epub ahead of print]
    The structural basis for inhibition of ribosomal translocation by viomycin.
    Ling Zhang, Ying-Hui Wang, Xing Zhang, Laura Lancaster, Jie Zhou, Harry F Noller.
      Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.
    Keywords:  ribosome; translocation; viomycin
    DOI:  https://doi.org/10.1073/pnas.2002888117
  25. J Immunol. 2020 May 01. pii: ji1901471. [Epub ahead of print]
    UHRF1 Controls Thymocyte Fate Decisions through the Epigenetic Regulation of EGR1 Expression.
    Yiwen Zhang, Yingshi Chen, Rong Ma, Yawen Jiang, Jun Liu, Yingtong Lin, Siqi Chen, Mengying Xia, Fan Zou, Junsong Zhang, Ting Pan, Lie Wang, Lai Wei, Hui Zhang.
      Thymocyte differentiation is a highly complex process that is accompanied by epigenetic changes. Ubiquitin-like containing PHD ring finger 1 (UHRF1) is a critical epigenetic modifier involved in various cellular processes. In this study, we demonstrated that it is highly expressed in T cell precursors of the thymus. Further, its deficiency results in significantly reduced thymocyte cellularity and thymus size in mice. Through systematic analysis based on single-cell RNA sequencing, we found that UHRF1 deficiency thwarts αβ T cell lineage development, whereas biasing γδ T lineage differentiation dampens the progression of immature single-positive cells. UHRF1 deficiency promotes the IL-17 secreting and RORγt expression in γδ T cell, indicating a Tγδ17 phenotype. Further, the analysis of gene-regulatory networks demonstrated that UHRF1 controls the expression of early growth response 1 (EGR1). UHRF1 interacts with DNA methyltransferase 1 (DNMT1) at the CpG promoter region of Egr1 loci and affects the nearby chromatin modifications of H3K9me3 and H3K4me3. Taken together, our results demonstrate that UHRF1 is a key factor that mediates the epigenetic regulation of EGR1 and, consequently, thymocyte fate decisions.
    DOI:  https://doi.org/10.4049/jimmunol.1901471
  26. Life Sci. 2020 Apr 23. pii: S0024-3205(20)30448-3. [Epub ahead of print]253 117700
    Regulation of the Golgi apparatus via GOLPH3-mediated new selective autophagy.
    Li-Qun Lu, Ming-Zhu Tang, Zhi-Hao Qi, Shi-Fang Huang, Yong-Qi He, Dian-Ke Li, Lan-Fang Li, Lin-Xi Chen.
      AIMS: Although previous studies elaborated that selective autophagy was involved in quality control of some organelles, including nucleus, mitochondria, the endoplasmic reticulum and peroxisomes, it remained unclear whether the selective autophagy of the Golgi apparatus (Golgiphagy) existed or not.MAIN METHODS: In this study, H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells were treated with autophagy inducers, Golgi stress inducers and cardiomyocytes hypertrophy stimulators. The Golgiphagy was evaluated by analysing the co-localization of Golgi markers and LC3B. Furthermore, the transmission electron microscope was used to observe the occurrence of Golgiphagy. The co-immunoprecipitation assay was used to evaluate the interaction of GOLPH3 and LC3B.
    KEY FINDINGS: Results showed that starvation promoted the co-localization of both GM130-positive and TGN46-positive Golgi fragments with LC3B-positive autophagosomes in H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells. Transmission electron microscopy images showed that Golgi apparatus was sequestered into the autophagosomes in the starvation group. Moreover, Golgi stress inducers also facilitated the co-localization of Golgi markers and LC3B in H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells. Furthermore, cardiomyocyte hypertrophy stimulators also triggered the appearance of Golgiphagy in H9c2 cells. Importantly, the co-immunoprecipitation assay indicated endogenous GOLPH3 interacted with LC3B in H9c2 cells, HUVECs, HA-VSMCs. However, knocking down GOLPH3 inhibited the Golgiphagy.
    SIGNIFICANCE: This study unveiled a new selective autophagy of the Golgi apparatus (Golgiphagy). In addition, GOLPH3 might act as a novel cargo receptor to regulate Golgiphagy. Maintaining homeostasis of the Golgi apparatus via GOLPH3-mediated autophagy was indispensable for cell survival.
    Keywords:  Autophagy; Cardiomyocytes hypertrophy; GOLPH3; Golgi apparatus; Golgi stress
    DOI:  https://doi.org/10.1016/j.lfs.2020.117700
  27. iScience. 2020 Apr 12. pii: S2589-0042(20)30235-2. [Epub ahead of print]23(5): 101050
    ER Stress Regulates Immunosuppressive Function of Myeloid Derived Suppressor Cells in Leprosy that Can Be Overcome in the Presence of IFN-γ.
    Kindra M Kelly-Scumpia, Aaron Choi, Roksana Shirazi, Hannah Bersabe, Esther Park, Philip O Scumpia, Maria T Ochoa, Jing Yu, Feiyang Ma, Matteo Pellegrini, Robert L Modlin.
      Myeloid derived suppressor cells (MDSCs) are a population of immature myeloid cells that suppress adaptive immune function, yet the factors that regulate their suppressive function in patients with infection remain unclear. We studied MDSCs in patients with leprosy, a disease caused by Mycobacterium leprae, where clinical manifestations present on a spectrum that correlate with immunity to the pathogen. We found that HLA-DR-CD33+CD15+ MDSCs were increased in blood from patients with disseminated/progressive lepromatous leprosy and possessed T cell-suppressive activity as compared with self-limiting tuberculoid leprosy. Mechanistically, we found ER stress played a critical role in regulating the T cell suppressive activity in these MDSCs. Furthermore, ER stress augmented IL-10 production, contributing to MDSC activity, whereas IFN-γ allowed T cells to overcome MDSC suppressive activity. These studies highlight a regulatory mechanism that links ER stress to IL-10 in mediating MDSC suppressive function in human infectious disease.
    Keywords:  Biological Sciences; Cell Biology; Functional Aspects of Cell Biology; Immune Response; Immunology
    DOI:  https://doi.org/10.1016/j.isci.2020.101050
  28. Nat Commun. 2020 May 01. 11(1): 2147
    Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly.
    Tanay Thakar, Wendy Leung, Claudia M Nicolae, Kristen E Clements, Binghui Shen, Anja-Katrin Bielinsky, George-Lucian Moldovan.
      Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.
    DOI:  https://doi.org/10.1038/s41467-020-16096-w
  29. EMBO J. 2020 Apr 30. e104939
    Behind closed gates - chaperones and charged residues determine protein fate.
    Margreet B Koopman, Stefan Gd Rüdiger.
      Charged residues flanking aggregation-prone regions play a role in protein folding and prevention of aggregation. In this issue of The EMBO Journal, Houben et al exploit the role of such charged gatekeepers in aggregation suppression and find that negative charges are more effective than positive ones. Strikingly, the prominent Hsp70 chaperone has a strong preference for the less effective, basic gate keepers. This implies co-adaptation of chaperone specificity and composition of protein sequences in evolution.
    DOI:  https://doi.org/10.15252/embj.2020104939
  30. PLoS Pathog. 2020 Apr 27. 16(4): e1008458
    Hyd ubiquitinates the NF-κB co-factor Akirin to operate an effective immune response in Drosophila.
    Alexandre Cammarata-Mouchtouris, Xuan-Hung Nguyen, Adrian Acker, François Bonnay, Akira Goto, Amir Orian, Marie-Odile Fauvarque, Michael Boutros, Jean-Marc Reichhart, Nicolas Matt.
      The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor κB (NF-κB) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys63 (K63)-linked polyubiquitination of the NF-κB cofactor Akirin was required for efficient binding of Akirin to the NF-κB transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-κB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-κB pathway for the activation of Interleukin 6 (IL6) transcription by IL-1β in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.
    DOI:  https://doi.org/10.1371/journal.ppat.1008458
  31. FEBS J. 2020 Apr 26.
    Synthetic post-translational modifications of elongation factor P using the ligase EpmA.
    Miriam Pfab, Pavel Kielkowski, Ralph Krafczyk, Wolfram Volkwein, Stephan A Sieber, Jürgen Lassak, Kirsten Jung.
      Canonically, tRNA synthetases charge tRNAs. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-β-lysine to the ε-amino group of lysine 34 of the translation elongation factor P (EF-P) in Escherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight non-canonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants on in vitro translation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-β-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.
    Keywords:  IF-5A; amber suppression; aminoacyl-tRNA synthetase; unnatural amino acids; β-amino acid
    DOI:  https://doi.org/10.1111/febs.15346
  32. EMBO J. 2020 Apr 27. e105012
    Derlins with scissors: primordial ERAD in bacteria.
    Julia D Knopf, Marius K Lemberg.
      Rhomboid intramembrane serine proteases are present in all kingdoms of life, but as we do not know their substrates in many species, it remains puzzling why rhomboids are among the most-conserved integral membrane proteins. Two new studies in The EMBO Journal by Began et al and Liu et al now link bacterial rhomboid proteases to membrane protein degradation, showing striking similarities to what is known about eukaryotic rhomboid family proteins, thus pointing toward a conserved membrane surveillance mechanism.
    DOI:  https://doi.org/10.15252/embj.2020105012
  33. Semin Cancer Biol. 2020 Apr 22. pii: S1044-579X(20)30085-7. [Epub ahead of print]
    Cullin-RING Ligase 5: Functional characterization and its role in human cancers.
    Yongchao Zhao, Xiufang Xiong, Yi Sun.
      Cullin-RING ligase 5 (CRL5) is a multi-protein complex and consists of a scaffold protien cullin 5, a RING protein RBX2 (also known as ROC2 or SAG), adaptor proteins Elongin B/C, and a substrate receptor protein SOCS. Through targeting a variety of substrates for proteasomal degradation or modulating various protein-protein interactions, CRL5 is involved in regulation of many biological processes, such as cytokine signal transduction, inflammation, viral infection, and oncogenesis. As many substrates of CRL5 are well-known oncoproteins or tumor suppressors, abnormal regulation of CRL5 is commonly found in human cancers. In this review, we first briefly introduce each of CRL5 components, and then discuss the biological processes regulated by four members of SOCS-box-containing substrate receptor family through substrate degradation. We next described how CRL5 is hijacked by a variety of viral proteins to degrade host anti-viral proteins, which facilitates virus infection. We further discuss the regulation of CUL5 and its various roles in human cancers, acting as either a tumor suppressor or an oncoprotein in a context-dependent manner. Finally, we propose novel insights for future perspectives on the validation of cullin-5 and other CRL5 components as potential targets, and possible targeting strategies to discover CRL5 inhibitors for anti-cancer and anti-virus therapies.
    Keywords:  CRL5; Cullin 5; RBX2/SAG; SOCS; tumorigenesis
    DOI:  https://doi.org/10.1016/j.semcancer.2020.04.003
  34. J Cell Physiol. 2020 Apr 26.
    Spliced X-box binding protein 1 induces liver cancer cell death via activating the Mst1-JNK-mROS signalling pathway.
    Jie Song, Wei Zhao, Chang Lu, Xue Shao.
      Previous studies have found that the primary pathogenesis of liver cancer progression is linked to excessive cancer cell proliferation and rapid metastasis. Although therapeutic advances have been made for the treatment of liver cancer, the mechanism underlying the liver cancer progression has not been fully addressed. In the present study, we explored the role of spliced X-box binding protein 1 (XBP1) in regulating the viability and death of liver cancer cells in vitro. Our study demonstrated that XBP1 was upregulated in liver cancer cells when compared to the primary hepatocytes. Interestingly, the deletion of XBP1 could reduce the viability of liver cancer cells in vitro via inducing apoptotic response. Further, we found that XBP1 downregulation was also linked to proliferation arrest and migration inhibition. At the molecular levels, XBP1 inhibition is followed by activation of the Mst1 pathway which promoted the phosphorylation of c-Jun N-terminal kinase (JNK). Then, the active Mst1-JNK pathway mediated mitochondrial reactive oxygen species (mROS) overproduction and then excessive ROS induced cancer cell death. Therefore, our study demonstrated a novel role played by XBP1 in modulating the viability of liver cancer cells via the Mst1-JNK-mROS pathways.
    Keywords:  Mst1 pathway; XBP1; apoptosis; liver cancer
    DOI:  https://doi.org/10.1002/jcp.29742
  35. EMBO Mol Med. 2020 Apr 29. e12075
    Cellular mechanisms and treatments for chemobrain: insight from aging and neurodegenerative diseases.
    Lien D Nguyen, Barbara E Ehrlich.
      Chemotherapy is a life-saving treatment for cancer patients, but also causes long-term cognitive impairment, or "chemobrain", in survivors. However, several challenges, including imprecise diagnosis criteria, multiple confounding factors, and unclear and heterogeneous molecular mechanisms, impede effective investigation of preventions and treatments for chemobrain. With the rapid increase in the number of cancer survivors, chemobrain is an urgent but unmet clinical need. Here, we leverage the extensive knowledge in various fields of neuroscience to gain insights into the mechanisms for chemobrain. We start by outlining why the post-mitotic adult brain is particularly vulnerable to chemotherapy. Next, through drawing comparisons with normal aging, Alzheimer's disease, and traumatic brain injury, we identify universal cellular mechanisms that may underlie the cognitive deficits in chemobrain. We further identify existing neurological drugs targeting these cellular mechanisms that can be repurposed as treatments for chemobrain, some of which were already shown to be effective in animal models. Finally, we briefly describe future steps to further advance our understanding of chemobrain and facilitate the development of effective preventions and treatments.
    Keywords:  aging; chemotherapy; cognitive impairment; neurodegenerative diseases; traumatic brain injury
    DOI:  https://doi.org/10.15252/emmm.202012075
  36. Stem Cells. 2020 Apr 28.
    N-glycosylation controls inflammatory licensing-triggered PD-L1 upregulation in human mesenchymal stromal cells.
    V Strauch, D Saul, M Berisha, A Mackensen, D Mougiakakos, R Jitschin.
      Mesenchymal stromal cells (MSCs) are characterized by their multipotency, regenerative potential, and immunoregulatory properties. Nowadays, MSCs represent a promising cell-therapeutic option for hyperinflammatory conditions such as graft-vs-host disease following allogeneic hematopoietic stem cell transplantation. A better understanding of their biology is a prerequisite for improving their treatment efficacy. Emerging evidence suggests that immunosuppressive properties are not constitutively active in MSCs. Instead, microenvironmental inflammatory stimuli such as the cytokines interferon (IFN)-γ or tumor necrosis factor (TNF)-α license MSCs to acquire a tolerance-promoting phenotype. The immunological checkpoint molecule programmed death-ligand 1 (PD-L1) is an important regulator of T-cell responses. Binding of PD-L1 to the programmed cell death protein 1 (PD-1) receptor on T cells suppresses their activation, proliferation, and induces apoptosis. Previous studies have revealed that cell surface expression and secretion of PD-L1 are part of the MSCs' immunomodulatory armamentarium. Here, we report that inflammatory licensing leads to an enhanced PD-L1 cell surface expression and secretion, which are both accompanied by an increased posttranslational protein N-glycosylation. These post-translational modifications have been shown to be critical for key biological processes such as cell trafficking, receptor signaling, and immunohomeostasis. In fact, promoting N-glycosylation in MSCs yielded increased PD-L1 levels. We report for the first time that PD-L1 N-glycosylation plays a decisive role for its transport to the MSCs' cell surface and its subsequent secretion (in response to proinflammatory trigger). Our data offer insights into a novel regulatory mechanism with the potential to be exploited as a means to foster the immunosuppressive potency of human MSCs. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: Mesenchymal stromal cells (MSCs) possess immunoregulatory properties and are hypoimmunogenic. These characteristics of MSCs make them promising candidates for cell therapeutic approaches in hyperinflammatory conditions such as graft-vs-host disease following allogeneic stem cell transplantation. This study shows that inflammatory stimuli promote expression and secretion of PD-L1 in human MSCs. It reveals for the first time that this adaptive process is accompanied by an enhanced N-glycosylation, which is a prerequisite for PD-L1 to be transported to the cell surface and to be secreted. Therefore, promoting N-glycosylation could be explored as a means to improve the MSCs' therapeutic efficacy.
    Keywords:  Immunometabolism; Immunoregulation; Mesenchymal Stromal Cells; N-glycosylation; PD-L1
    DOI:  https://doi.org/10.1002/stem.3190
  37. J Cell Biol. 2020 Jul 06. pii: e201910131. [Epub ahead of print]219(7):
    A modified lysosomal organelle mediates nonlytic egress of reovirus.
    Isabel Fernández de Castro, Raquel Tenorio, Paula Ortega-González, Jonathan J Knowlton, Paula F Zamora, Christopher H Lee, José J Fernández, Terence S Dermody, Cristina Risco.
      Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1-positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from ∼4.5-5 to ∼6.1 after recruitment to VIs and before incorporation of virions. ET of VI-SO-MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention.
    DOI:  https://doi.org/10.1083/jcb.201910131
  38. Life Sci Alliance. 2020 Jun;pii: e202000735. [Epub ahead of print]3(6):
    Caspase-1 cleaves Bid to release mitochondrial SMAC and drive secondary necrosis in the absence of GSDMD.
    Rosalie Heilig, Marisa Dilucca, Dave Boucher, Kaiwen W Chen, Dora Hancz, Benjamin Demarco, Kateryna Shkarina, Petr Broz.
      Caspase-1 drives a lytic inflammatory cell death named pyroptosis by cleaving the pore-forming cell death executor gasdermin-D (GSDMD). Gsdmd deficiency, however, only delays cell lysis, indicating that caspase-1 controls alternative cell death pathways. Here, we show that in the absence of GSDMD, caspase-1 activates apoptotic initiator and executioner caspases and triggers a rapid progression into secondary necrosis. GSDMD-independent cell death required direct caspase-1-driven truncation of Bid and generation of caspase-3 p19/p12 by either caspase-8 or caspase-9. tBid-induced mitochondrial outer membrane permeabilization was also required to drive SMAC release and relieve inhibitor of apoptosis protein inhibition of caspase-3, thereby allowing caspase-3 auto-processing to the fully active p17/p12 form. Our data reveal that cell lysis in inflammasome-activated Gsdmd-deficient cells is caused by a synergistic effect of rapid caspase-1-driven activation of initiator caspases-8/-9 and Bid cleavage, resulting in an unusually fast activation of caspase-3 and immediate transition into secondary necrosis. This pathway might be advantageous for the host in counteracting pathogen-induced inhibition of GSDMD but also has implications for the use of GSDMD inhibitors in immune therapies for caspase-1-dependent inflammatory disease.
    DOI:  https://doi.org/10.26508/lsa.202000735
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