bims-proteo Biomed News
on Proteostasis
Issue of 2020–06–07
fifty-two papers selected by
Eric Chevet, INSERM
  1. Chaperone-Mediated Protein Disaggregation Triggers Proteolytic Clearance of Intra-nuclear Protein Inclusions.
  2. Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain.
  3. Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus.
  4. BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.
  5. Molecular chaperone Hsp90 regulates heterochromatin assembly through stabilizing multiple complexes in fission yeast.
  6. Furin-instructed molecular self-assembly actuates endoplasmic reticulum stress-mediated apoptosis for cancer therapy.
  7. Functional coupling of presequence processing and degradation in human mitochondria.
  8. The ER stress-autophagy axis: implications for cognitive dysfunction in diabetes mellitus.
  9. Mechanisms of aortic carboxypeptidase-like protein secretion and identification of an intracellularly retained variant associated with Ehlers-Danlos syndrome.
  10. Foot-and-mouth disease virus induces PERK mediated autophagy to suppress antiviral interferon response.
  11. CRISPR screen in regulatory T cells reveals modulators of Foxp3.
  12. Secretory Autophagy and Its Relevance in Metabolic and Degenerative Disease.
  13. Flightless-1 inhibits ER stress-induced apoptosis in colorectal cancer cells by regulating Ca2+ homeostasis.
  14. Lem2 and Lnp1 maintain the membrane boundary between the nuclear envelope and endoplasmic reticulum.
  15. Selective autophagy controls the stability of transcription factor IRF3 to balance type I interferon production and immune suppression.
  16. Rescue of HSP70 in Spinal Neurons Alleviates Opioids-Induced Hyperalgesia via the Suppression of Endoplasmic Reticulum Stress in Rodents.
  17. IGF1R is an entry receptor for respiratory syncytial virus.
  18. Mechanisms and Regulation of RNA Condensation in RNP Granule Formation.
  19. Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA.
  20. The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K.
  21. MARCH5 mediates NOXA-dependent MCL1 degradation driven by kinase inhibitors and integrated stress response activation.
  22. A SAM-I riboswitch with the ability to sense and respond to uncharged initiator tRNA.
  23. Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components.
  24. Break on Through: Golgi-Derived Vesicles Aid in Mitochondrial Fission.
  25. Structure of the ER membrane complex, a transmembrane-domain insertase.
  26. Ritonavir and Lopinavir Suppress RCE1 and CAAX Rab Proteins Sensitizing the Liver to Organelle Stress and Injury.
  27. Olig2 SUMOylation protects against genotoxic damage response by antagonizing p53 gene targeting.
  28. KCTD10 Biology: An Adaptor for the Ubiquitin E3 Complex Meets Multiple Substrates: Emerging Divergent Roles of the cullin-3/KCTD10 E3 Ubiquitin Ligase Complex in Various Cell Lines.
  29. Development of Resistance to Endoplasmic Reticulum Stress-Inducing Agents in Mouse Leukemic L1210 Cells.
  30. Transcription factors activated through RIP (Regulated Intramembrane Proteolysis) and RAT (Regulated Alternative Translocation)).
  31. Valosin-Containing Protein, a Calcium-Associated ATPase Protein, in Endoplasmic Reticulum and Mitochondrial Function and Its Implications for Diseases.
  32. Src couples estrogen receptor to the anticipatory unfolded protein response and regulates cancer cell fate under stress.
  33. O-GlcNAc transferase promotes influenza A virus-induced cytokine storm by targeting interferon regulatory factor-5.
  34. Enhanced phosphorylation of PERK in primary cultured neurons as an autonomous neuronal response to prion infection.
  35. Role of the lipid bilayer in outer membrane protein folding in Gram negative bacteria.
  36. NOD1/NOD2 and RIP2 Regulate Endoplasmic Reticulum Stress-Induced Inflammation during Chlamydia Infection.
  37. A-kinase-anchoring protein 1 (dAKAP1)-based signaling complexes coordinate local protein synthesis at the mitochondrial surface.
  38. The Coupling of Translational Control and Stress Responses.
  39. Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.
  40. Light microscopy of the endoplasmic reticulum-membrane contact sites in plants.
  41. The Regulatory Role of Reticulons in Neurodegeneration: Insights Underpinning Therapeutic Potential for Neurodegenerative Diseases.
  42. ER-phagy: Eating the Factory.
  43. Autophagy coupled to translation is required for long-term memory.
  44. Endoplasmic reticulum stress mediated apoptosis via JNK in MWCNT-exposed in vitro systems: size, surface functionalization and cell type specificity.
  45. HSF1 Activation Can Restrict HIV Replication.
  46. Proteasome subunit PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress.
  47. Parkin ubiquitinates phosphoglycerate dehydrogenase to suppress serine synthesis and tumor progression.
  48. ERdj8 governs the size of autophagosomes during the formation process.
  49. A Critical Role for ISGylation, Ubiquitination and, SUMOylation in Brain Damage: Implications for Neuroprotection.
  50. Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~ubiquitin conformation.
  51. E3 ubiquitin ligase ATL61 acts as a positive regulator in abscisic acid mediated drought response in Arabidopsis.
  52. UBE2T promotes glioblastoma invasion and migration via stabilizing GRP78 and regulating EMT.
  1. Cell Rep. 2020 Jun 02. pii: S2211-1247(20)30633-1. [Epub ahead of print]31(9): 107680
    Chaperone-Mediated Protein Disaggregation Triggers Proteolytic Clearance of Intra-nuclear Protein Inclusions.
    Fabian den Brave, Lucas V Cairo, Chandhuru Jagadeesan, Carmen Ruger-Herreros, Axel Mogk, Bernd Bukau, Stefan Jentsch.
      The formation of insoluble inclusions in the cytosol and nucleus is associated with impaired protein homeostasis and is a hallmark of several neurodegenerative diseases. Due to the absence of the autophagic machinery, nuclear protein aggregates require a solubilization step preceding degradation by the 26S proteasome. Using yeast, we identify a nuclear protein quality control pathway required for the clearance of protein aggregates. The nuclear J-domain protein Apj1 supports protein disaggregation together with Hsp70 but independent of the canonical disaggregase Hsp104. Disaggregation mediated by Apj1/Hsp70 promotes turnover rather than refolding. A loss of Apj1 activity uncouples disaggregation from proteasomal turnover, resulting in accumulation of toxic soluble protein species. Endogenous substrates of the Apj1/Hsp70 pathway include both nuclear and cytoplasmic proteins, which aggregate inside the nucleus upon proteotoxic stress. These findings demonstrate the coordinated activity of the Apj1/Hsp70 disaggregation system with the 26S proteasome in facilitating the clearance of toxic inclusions inside the nucleus.
    Keywords:  Apj1; Chaperone; Disaggregation; Hsp110; Hsp40; Hsp70; Proteostasis; nucleus; protein degradation
    DOI:  https://doi.org/10.1016/j.celrep.2020.107680
  2. J Biol Chem. 2020 Jun 01. pii: jbc.RA120.014071. [Epub ahead of print]
    Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain.
    Ngoc-Duc Doan, Azade S Hosseini, Agata A Bikovtseva, Michelle S Huang, Andrew S DiChiara, Louis J Papa, Antonius Koller, Matthew D Shoulders.
      Intracellular collagen assembly begins with the oxidative folding of ~30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Herein, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that "C-Pro-only" constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.
    Keywords:  ER quality control; Procollagen; collagen; extracellular matrix; mass spectrometry-based interactome; protein folding; protein quality control; protein secretion; proteostasis; triple-helix assembly
    DOI:  https://doi.org/10.1074/jbc.RA120.014071
  3. mBio. 2020 Jun 02. pii: e01060-20. [Epub ahead of print]11(3):
    Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus.
    Martin Weichert, José Guirao-Abad, Vishukumar Aimanianda, Karthik Krishnan, Christina Grisham, Patrick Snyder, Alex Sheehan, Ruthvik R Abbu, Hong Liu, Scott G Filler, Eric I Gruenstein, Jean-Paul Latgé, David S Askew.
      Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity.IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.
    Keywords:  Aspergillus fumigatus ; ER stress; HacA; SERCA; SPCA; UPR; calcium; cell wall; galactomannan
    DOI:  https://doi.org/10.1128/mBio.01060-20
  4. PLoS Genet. 2020 Jun 05. 16(6): e1008829
    BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.
    Timothy P Cheung, Jun-Yong Choe, Janet E Richmond, Hongkyun Kim.
      Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.
    DOI:  https://doi.org/10.1371/journal.pgen.1008829
  5. J Cell Sci. 2020 Jun 04. pii: jcs.244863. [Epub ahead of print]
    Molecular chaperone Hsp90 regulates heterochromatin assembly through stabilizing multiple complexes in fission yeast.
    Li Sun, Xiao-Min Liu, Wen-Zhu Li, Yuan-Yuan Yi, Xiangwei He, Yamei Wang, Quan-Wen Jin.
      In the fission yeast Schizosaccharomyces pombe, both RNAi machinery and RNAi- independent factors mediate transcriptional and posttranscriptional silencing and heterochromatin formation. Here, we show that the silencing of reporter genes at major native heterochromatic loci (centromeres, telomeres, mating-type locus and rDNA regions) and an artificially induced heterochromatin locus is alleviated in a fission yeast hsp90 mutant, hsp90-G84C Also, H3K9me2 enrichment at heterochromatin regions, especially at the mating-type locus and subtelomeres, is compromised, suggesting heterochromatin assembly defects. We further discovered that Hsp90 is required for stabilization or assembly of the RNAi effector complexes RITS and ARC, RNAi-independent factor Fft3, shelterin complex subunit Poz1 and SHREC. Our ChIP data suggest that Hsp90 regulates the efficient recruitment of CLRC by shelterin to chromosome ends and targeting of SHREC and Fft3 to mating type locus and/or rDNA region. Finally, our genetic analyses demonstrated that increased heterochromatin spreading restores silencing at subtelomeres in hsp90-G84C mutant. Thus, this work uncovers a conserved factor critical for promoting RNAi-dependent and -independent heterochromatin assembly and gene silencing through stabilizing multiple effectors and effector complexes.
    Keywords:  Fission yeast; Heat-shock molecular chaperon; Heterochromatic gene silencing; Hsp90; RNAi; Schizosaccharomyces pombe
    DOI:  https://doi.org/10.1242/jcs.244863
  6. Nanoscale. 2020 Jun 02.
    Furin-instructed molecular self-assembly actuates endoplasmic reticulum stress-mediated apoptosis for cancer therapy.
    Chenxing Fu, Jie Zhan, Junqi Huai, Shaodan Ma, Minghui Li, Guoqin Chen, Minsheng Chen, Yanbin Cai, Caiwen Ou.
      Protein quality control and proteostasis are essential to maintain cell survival as once disordered, they will trigger endoplasmic reticulum (ER) stress and even initiate apoptosis. Severe ER stress-mediated apoptosis is the cause of neurodegenerative diseases and expected to be a new target for cancer therapy. In this study, we designed a small molecule of 1-Nap to execute furin-instructed molecular self-assembly for selectively inhibiting the growth of MDA-MB-468 cells in vitro and in vivo. According to the results of transmission electron microscopy (TEM) and HPLC tracing analysis, 1-Nap is capable of self-assembling upon furin-instructed cleavage that transforms 1-Nap nanoparticles to 1-Nap nanofibers. Fluorescence imaging and Western-blot analysis results indicate that the furin-instructed self-assembly of 1-Nap rather than its ER-targeting interaction is indispensable for the ER stress and activation of apoptosis. The furin-instructed self-assembly of 1-Nap is associated with both the ER (1-Nap's targeting location) and the trans-Golgi network (furin's location); this inspired us to reasonably believe that the blocking of ER-to-Golgi traffic in the secretory pathway by molecular self-assembly may be the intrinsic motivation for controlling cell fate. This work provides a new way for the targeted disturbance of the proteostasis of cells through molecular self-assembly for developing cancer therapeutics.
    DOI:  https://doi.org/10.1039/d0nr00151a
  7. FEBS J. 2020 Jun 03.
    Functional coupling of presequence processing and degradation in human mitochondria.
    Cansu Kücükköse, Asli Aras Taskin, Adinarayana Marada, Tilman Brummer, Sven Dennerlein, Friederike-Nora Vögtle.
      The mitochondrial proteome is built and maintained mainly by import of nuclear-encoded precursor proteins. Most of these precursors use N-terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PrePR183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome.
    Keywords:  Alzheimer's disease; integrated stress response; mitochondrial proteostasis; precursor protein import; presequence degradation
    DOI:  https://doi.org/10.1111/febs.15358
  8. Clin Sci (Lond). 2020 Jun 12. 134(11): 1255-1258
    The ER stress-autophagy axis: implications for cognitive dysfunction in diabetes mellitus.
    Qingzhang Zhu.
      Unfolded protein response (UPR) often coordinates with autophagy to maintain cellular proteostasis. Disturbance of proteostasis correlates with diseases including diabetes and neurological complications. In a recent article in Clinical Science, Kong et al. highlighted the critical role of endoplasmic reticulum (ER) stress-autophagy axis in maintaining cognitive functions and provided pharmacological evidence with respect to cognitive improvements in a diabetic mouse model. These novel findings present new insights into the pathological mechanisms and therapeutic implications with the ER stress modulators in diabetes-related cognitive dysfunction.
    Keywords:  ER stress; autophagy; cognitive dysfunction; diabetes
    DOI:  https://doi.org/10.1042/CS20200235
  9. J Biol Chem. 2020 Jun 01. pii: jbc.RA120.013902. [Epub ahead of print]
    Mechanisms of aortic carboxypeptidase-like protein secretion and identification of an intracellularly retained variant associated with Ehlers-Danlos syndrome.
    Neya Vishwanath, William J Monis, Gwendolyn A Hoffmann, Bhavana Ramachandran, Vincent DiGiacomo, Joyce Y Wong, Michael L Smith, Matthew D Layne.
      Aortic carboxypeptidase-like protein (ACLP) is a collagen-binding extracellular matrix (ECM) protein that has important roles in wound healing and fibrosis. ACLP contains thrombospondin repeats, a collagen-binding discoidin domain, and a catalytically inactive metallocarboxypeptidase domain. Recently, mutations in the ACLP-encoding gene, AE-binding protein 1 (AEBP1), have been discovered, leading to the identification of a new variant of Ehlers-Danlos syndrome (EDS) causing connective tissue disruptions in multiple organs. Currently, little is known about the mechanisms of ACLP secretion or the role of posttranslational modifications in these processes. We show here that the secreted form of ACLP contains N-linked glycosylation and that inhibition of glycosylation results in its intracellular retention. Using site-directed mutagenesis, we determined that glycosylation of Asn-471 and Asn-1030 is necessary for ACLP secretion and identified a specific N-terminal proteolytic ACLP fragment. To determine the contribution of secreted ACLP to ECM mechanical properties, we generated and mechanically tested wet-spun collagen ACLP composite fibers, finding that ACLP enhances the modulus (or stiffness), toughness, and tensile strength of the fibers. Some AEBP1 mutations were null alleles, whereas others resulted in expressed proteins. We tested the hypothesis that a recently discovered 40-amino-acid mutation and insertion in the ACLP discoidin domain regulates collagen binding and assembly. Interestingly, we found that this protein variant is retained intracellularly and induces endoplasmic reticulum (ER) stress identified with an XBP1-based ER stress reporter. Our findings highlight the importance of N-linked glycosylation of ACLP for its secretion and contribute to our understanding of ACLP-dependent disease pathologies.
    Keywords:  Ehlers-Danlos syndrome; aortic carboxypeptidase-like protein (ACLP); collagen; endoplasmic reticulum stress (ER stress); extracellular matrix (ECM); genetic disease; glycoprotein secretion; glycosylation; post-translational modification (PTM)
    DOI:  https://doi.org/10.1074/jbc.RA120.013902
  10. J Cell Sci. 2020 Jun 01. pii: jcs.240622. [Epub ahead of print]
    Foot-and-mouth disease virus induces PERK mediated autophagy to suppress antiviral interferon response.
    H B Ranjitha, Veena Ammanathan, Neha Guleria, Madhusudan Hosamani, B P Sreenivasa, V V Dhanesh, Rashmi Santhoshkumar, B K Chandrasekhar Sagar, B P Mishra, R K Singh, Aniket Sanyal, Ravi Manjithaya, Suresh H Basagoudanavar.
      Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in cloven-hoofed animals. FMDV replication associated viral protein expression induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR), in turn inducing autophagy to restore cellular homeostasis. We observed that inhibition of BiP, a master regulator of ER stress and UPR, decreased FMDV infection confirming their involvement. Further, we show that the FMDV infection induces UPR mainly through PKR-like ER kinase (PERK)-mediated pathway. Knockdown of PERK and chemical inhibition of PERK activation resulted in decreased expression of FMDV proteins along with the reduction of autophagy marker protein LC3B-II. There are conflicting reports on the role of autophagy in FMDV multiplication. Our study systematically demonstrates that during FMDV infection, PERK mediated UPR stimulated an increased level of endogenous LC3B-II and turnover of SQSTM1, thus confirming the activation of functional autophagy. Modulation of UPR and autophagy by pharmacological and genetic approaches resulted in reduced viral progeny, by enhancing antiviral interferon response. Taken together, this study underscores the prospect of exploring the PERK mediated autophagy as an antiviral target.
    Keywords:  Autophagy; Foot-and-mouth disease virus; Interferon; LC3; P-eIF2α; PERK; Unfolded protein response
    DOI:  https://doi.org/10.1242/jcs.240622
  11. Nature. 2020 Apr 29.
    CRISPR screen in regulatory T cells reveals modulators of Foxp3.
    Jessica T Cortez, Elena Montauti, Eric Shifrut, Jovylyn Gatchalian, Yusi Zhang, Oren Shaked, Yuanming Xu, Theodore L Roth, Dimitre R Simeonov, Yana Zhang, Siqi Chen, Zhongmei Li, Jonathan M Woo, Josephine Ho, Ian A Vogel, Grace Y Prator, Bin Zhang, Youjin Lee, Zhaolin Sun, Igal Ifergan, Frédéric Van Gool, Diana C Hargreaves, Jeffrey A Bluestone, Alexander Marson, Deyu Fang.
      Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.
    DOI:  https://doi.org/10.1038/s41586-020-2246-4
  12. Front Endocrinol (Lausanne). 2020 ;11 266
    Secretory Autophagy and Its Relevance in Metabolic and Degenerative Disease.
    Claudio Daniel Gonzalez, Roxana Resnik, Maria Ines Vaccaro.
      Proteins to be secreted through so-called "conventional mechanisms" are characterized by the presence of an N-terminal peptide that is a leader or signal peptide, needed for access to the endoplasmic reticulum and the Golgi apparatus for further secretion. However, some relevant cytosolic proteins lack of this signal peptides and should be secreted by different unconventional or "non-canonical" processes. One form of this unconventional secretion was named secretory autophagy (SA) because it is specifically associated with the autophagy pathway. It is defined by ATG proteins that regulate the biogenesis of the autophagosome, its representative organelle. The canonical macroautophagy involves the fusion of the autophagosomes with lysosomes for content degradation, whereas the SA pathway bypasses this degradative process to allow the secretion. ATG5, as well as other factors involved in autophagy such as BCN1, are also activated as part of the secretory pathway. SA has been recognized as a new mechanism that is becoming of increasing relevance to explain the unconventional secretion of a series of cytosolic proteins that have critical biological importance. Also, SA may play a role in the release of aggregation-prone protein since it has been related to the autophagosome biogenesis machinery. SA requires the autophagic pathway and both, secretory autophagy and canonical degradative autophagy are at the same time, integrated and highly regulated processes that interact in ultimate cross-talking molecular mechanisms. The potential implications of alterations in SA, its cargos, pathways, and regulation in human diseases such as metabolic/aging pathological processes are predictable. Further research of SA as potential target of therapeutic intervention is deserved.
    Keywords:  ATG (autophagy-related) proteins; IL-1β; aggregate-prone proteins; macroautophagy; unconventional protein secretion
    DOI:  https://doi.org/10.3389/fendo.2020.00266
  13. Exp Mol Med. 2020 Jun 05.
    Flightless-1 inhibits ER stress-induced apoptosis in colorectal cancer cells by regulating Ca2+ homeostasis.
    Sun Sil Choi, Sang Kwon Lee, Joong Kwan Kim, Hye-Kyung Park, Eujin Lee, Jinho Jang, Yo Han Lee, Keon Woo Khim, Ji-Min Hyun, Hye-Jin Eom, Semin Lee, Byuong Heon Kang, Young Chan Chae, Kyungjae Myung, Seung-Jae Myung, Chan Young Park, Jang Hyun Choi.
      The endoplasmic reticulum (ER) stress response is an adaptive mechanism that is activated upon disruption of ER homeostasis and protects the cells against certain harmful environmental stimuli. However, critical and prolonged cell stress triggers cell death. In this study, we demonstrate that Flightless-1 (FliI) regulates ER stress-induced apoptosis in colon cancer cells by modulating Ca2+ homeostasis. FliI was highly expressed in both colon cell lines and colorectal cancer mouse models. In a mouse xenograft model using CT26 mouse colorectal cancer cells, tumor formation was slowed due to elevated levels of apoptosis in FliI-knockdown (FliI-KD) cells. FliI-KD cells treated with ER stress inducers, thapsigargin (TG), and tunicamycin exhibited activation of the unfolded protein response (UPR) and induction of UPR-related gene expression, which eventually triggered apoptosis. FliI-KD increased the intracellular Ca2+ concentration, and this upregulation was caused by accelerated ER-to-cytosolic efflux of Ca2+. The increase in intracellular Ca2+ concentration was significantly blocked by dantrolene and tetracaine, inhibitors of ryanodine receptors (RyRs). Dantrolene inhibited TG-induced ER stress and decreased the rate of apoptosis in FliI-KD CT26 cells. Finally, we found that knockdown of FliI decreased the levels of sorcin and ER Ca2+ and that TG-induced ER stress was recovered by overexpression of sorcin in FliI-KD cells. Taken together, these results suggest that FliI regulates sorcin expression, which modulates Ca2+ homeostasis in the ER through RyRs. Our findings reveal a novel mechanism by which FliI influences Ca2+ homeostasis and cell survival during ER stress.
    DOI:  https://doi.org/10.1038/s12276-020-0448-3
  14. Commun Biol. 2020 Jun 01. 3(1): 276
    Lem2 and Lnp1 maintain the membrane boundary between the nuclear envelope and endoplasmic reticulum.
    Yasuhiro Hirano, Yasuha Kinugasa, Hiroko Osakada, Tomoko Shindo, Yoshino Kubota, Shinsuke Shibata, Tokuko Haraguchi, Yasushi Hiraoka.
      The nuclear envelope (NE) continues to the endoplasmic reticulum (ER). Proper partitioning of NE and ER is crucial for cellular activity, but the key factors maintaining the boundary between NE and ER remain to be elucidated. Here we show that the conserved membrane proteins Lem2 and Lnp1 cooperatively play a crucial role in maintaining the NE-ER membrane boundary in fission yeast Schizosaccharomyces pombe. Cells lacking both Lem2 and Lnp1 caused severe growth defects associated with aberrant expansion of the NE/ER membranes, abnormal leakage of nuclear proteins, and abnormal formation of vacuolar-like structures in the nucleus. Overexpression of the ER membrane protein Apq12 rescued the growth defect associated with membrane disorder caused by the loss of Lem2 and Lnp1. Genetic analysis showed that Apq12 had overlapping functions with Lnp1. We propose that a membrane protein network with Lem2 and Lnp1 acts as a critical factor to maintain the NE-ER boundary.
    DOI:  https://doi.org/10.1038/s42003-020-0999-9
  15. Autophagy. 2020 May 31. 1-14
    Selective autophagy controls the stability of transcription factor IRF3 to balance type I interferon production and immune suppression.
    Yaoxing Wu, Shouheng Jin, Qingxiang Liu, Yu Zhang, Ling Ma, Zhiyao Zhao, Shuai Yang, Yi-Ping Li, Jun Cui.
      IRF3 (interferon regulatory factor 3) is one of the most critical transcription factors in antiviral innate immune signaling, which is ubiquitously expressed in a variety of cells. Although it has been demonstrated that IRF3 can provoke multiple cellular processes during viral infection, including type I interferon (IFN) production, the mechanisms underlying the precise regulation of IRF3 activity are still not completely understood. Here, we report that selective macroautophagy/autophagy mediated by cargo receptor CALCOCO2/NDP52 promotes the degradation of IRF3 in a virus load-dependent manner. Deubiquitinase PSMD14/POH1 prevents IRF3 from autophagic degradation by cleaving the K27-linked poly-ubiquitin chains at lysine 313 on IRF3 to maintain its basal level and IRF3-mediated type I IFN activation. The autophagic degradation of IRF3 mediated by PSMD14 or CALCOCO2 ensures the precise control of IRF3 activity and fine-tunes the immune response against viral infection. Our study reveals the regulatory role of PSMD14 in balancing IRF3-centered IFN activation with immune suppression and provides insights into the crosstalk between selective autophagy and type I IFN signaling.
    ABBREVIATIONS: ATG5: autophagy related gene 5; Baf A1: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CGAS: cyclic GMP-AMP synthase; DDX58/RIG-I: DExD/H-box helicase 58; DUBs: deubiquitinating enzymes; IFN: interferon; IRF3: interferon regulatory factor 3; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMPs: pathogen-associated molecule patterns; PBMC: peripheral blood mononuclear cell; PSMD14/POH1: proteasome 26S subunit, non-ATPase 14; RIPA: RLR-induced IRF3-mediated pathway of apoptosis; SeV: Sendai virus; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; Ub: ubiquitin; WT: wild type.
    Keywords:  Antiviral immunity; deubiquitination; immune suppression; selective autophagy; type I interferon signaling
    DOI:  https://doi.org/10.1080/15548627.2020.1761653
  16. Front Cell Dev Biol. 2020 ;8 269
    Rescue of HSP70 in Spinal Neurons Alleviates Opioids-Induced Hyperalgesia via the Suppression of Endoplasmic Reticulum Stress in Rodents.
    Tong-Tong Lin, Jie Qu, Chao-Yu Wang, Xing Yang, Fan Hu, Liang Hu, Xue-Feng Wu, Chun-Yi Jiang, Wen-Tao Liu, Yuan Han.
      A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opioids. Endoplasmic reticulum (ER) stress has been shown to contribute to neuropathic or inflammatory pain, but its roles in opioids-induced hyperalgesia (OIH) are elusive. Here, we provide the first direct evidence that ER stress is a significant driver of OIH. GRP78, the ER stress marker, is markedly upregulated in neurons in the spinal cord after chronic morphine treatment. At the same time, morphine induces the activation of three arms of unfolded protein response (UPR): inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1), protein kinase RNA-like ER kinase/eukaryotic initiation factor 2 subunit alpha (PERK/eIF2α), and activating transcription factor 6 (ATF6). Notably, we found that inhibition on either IRE1α/XBP1 or ATF6, but not on PERK/eIF2α could attenuate the development of OIH. Consequently, ER stress induced by morphine enhances PKA-mediated phosphorylation of NMDA receptor subunit 1(NR1) and leads to OIH. We further showed that heat shock protein 70 (HSP70), a molecular chaperone involved in protein folding in ER, is heavily released from spinal neurons after morphine treatment upon the control of KATP channel. Glibenclamide, a classic KATP channel blocker that inhibits the efflux of HSP70 from cytoplasm to extracellular environment, or HSP70 overexpression in neurons, could markedly suppress morphine-induced ER stress and hyperalgesia. Taken together, our findings uncover the induction process and the central role of ER stress in the development of OIH and support a novel strategy for anti-OIH treatment.
    Keywords:   NR1; HSP70; PKA; endoplasmic reticulum stress; opioids-induced hyperalgesia; unfolded protein response
    DOI:  https://doi.org/10.3389/fcell.2020.00269
  17. Nature. 2020 Jun 03.
    IGF1R is an entry receptor for respiratory syncytial virus.
    Cameron D Griffiths, Leanne M Bilawchuk, John E McDonough, Kyla C Jamieson, Farah Elawar, Yuchen Cen, Wenming Duan, Cindy Lin, Haeun Song, Jean-Laurent Casanova, Steven Ogg, Lionel Dylan Jensen, Bernard Thienpont, Anil Kumar, Tom C Hobman, David Proud, Theo J Moraes, David J Marchant.
      Pneumonia resulting from infection is one of the leading causes of death worldwide. Pulmonary infection by the respiratory syncytial virus (RSV) is a large burden on human health, for which there are few therapeutic options1. RSV targets ciliated epithelial cells in the airways, but how viruses such as RSV interact with receptors on these cells is not understood. Nucleolin is an entry coreceptor for RSV2 and also mediates the cellular entry of influenza, the parainfluenza virus, some enteroviruses and the bacterium that causes tularaemia3,4. Here we show a mechanism of RSV entry into cells in which outside-in signalling, involving binding of the prefusion RSV-F glycoprotein with the insulin-like growth factor-1 receptor, triggers the activation of protein kinase C zeta (PKCζ). This cellular signalling cascade recruits nucleolin from the nuclei of cells to the plasma membrane, where it also binds to RSV-F on virions. We find that inhibiting PKCζ activation prevents the trafficking of nucleolin to RSV particles on airway organoid cultures, and reduces viral replication and pathology in RSV-infected mice. These findings reveal a mechanism of virus entry in which receptor engagement and signal transduction bring the coreceptor to viral particles at the cell surface, and could form the basis of new therapeutics to treat RSV infection.
    DOI:  https://doi.org/10.1038/s41586-020-2369-7
  18. Trends Biochem Sci. 2020 May 11. pii: S0968-0004(20)30121-3. [Epub ahead of print]
    Mechanisms and Regulation of RNA Condensation in RNP Granule Formation.
    Devin Tauber, Gabriel Tauber, Roy Parker.
      Ribonucleoprotein (RNP) granules are RNA-protein assemblies that are involved in multiple aspects of RNA metabolism and are linked to memory, development, and disease. Some RNP granules form, in part, through the formation of intermolecular RNA-RNA interactions. In vitro, such trans RNA condensation occurs readily, suggesting that cells require mechanisms to modulate RNA-based condensation. We assess the mechanisms of RNA condensation and how cells modulate this phenomenon. We propose that cells control RNA condensation through ATP-dependent processes, static RNA buffering, and dynamic post-translational mechanisms. Moreover, perturbations in these mechanisms can be involved in disease. This reveals multiple cellular mechanisms of kinetic and thermodynamic control that maintain the proper distribution of RNA molecules between dispersed and condensed forms.
    Keywords:  DEAD-box protein; P-body; RNA chaperone; RNA-binding protein; RNA–RNA interaction; biomolecular condensate; ribonucleoprotein granule; stress granule
    DOI:  https://doi.org/10.1016/j.tibs.2020.05.002
  19. EMBO J. 2020 Jun 03. e104096
    Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA.
    Szymon W Kmiecik, Laura Le Breton, Matthias P Mayer.
      The heat shock response is a universal transcriptional response to proteotoxic stress orchestrated by heat shock transcription factor Hsf1 in all eukaryotic cells. Despite over 40 years of intense research, the mechanism of Hsf1 activity regulation remains poorly understood at the molecular level. In metazoa, Hsf1 trimerizes upon heat shock through a leucine-zipper domain and binds to DNA. How Hsf1 is dislodged from DNA and monomerized remained enigmatic. Here, using purified proteins, we demonstrate that unmodified trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1. Hsc70 binds to multiple sites in Hsf1 with different affinities. Hsf1 trimers are monomerized by successive cycles of entropic pulling, unzipping the triple leucine-zipper. Starting this unzipping at several protomers of the Hsf1 trimer results in faster monomerization. This process directly monitors the concentration of Hsc70 and DnaJB1. During heat shock adaptation, Hsc70 first binds to a high-affinity site in the transactivation domain, leading to partial attenuation of the response, and subsequently, at higher concentrations, Hsc70 removes Hsf1 from DNA to restore the resting state.
    Keywords:  DNA binding; Hsp70; attenuation; entropic pulling; heat shock response
    DOI:  https://doi.org/10.15252/embj.2019104096
  20. Nature. 2020 Jun 03.
    The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K.
    Mikołaj Słabicki, Zuzanna Kozicka, Georg Petzold, Yen-Der Li, Manisha Manojkumar, Richard D Bunker, Katherine A Donovan, Quinlan L Sievers, Jonas Koeppel, Dakota Suchyta, Adam S Sperling, Emma C Fink, Jessica A Gasser, Li R Wang, Steven M Corsello, Rob S Sellar, Max Jan, Dennis Gillingham, Claudia Scholl, Stefan Fröhling, Todd R Golub, Eric S Fischer, Nicolas H Thomä, Benjamin L Ebert.
      Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.
    DOI:  https://doi.org/10.1038/s41586-020-2374-x
  21. Elife. 2020 Jun 02. pii: e54954. [Epub ahead of print]9
    MARCH5 mediates NOXA-dependent MCL1 degradation driven by kinase inhibitors and integrated stress response activation.
    Seiji Arai, Andreas Varkaris, Mannan Nouri, Sen Chen, Lisha Xie, Steven P Balk.
      MCL1 has critical antiapoptotic functions and its levels are tightly regulated by ubiquitylation and degradation, but mechanisms that drive this degradation, particularly in solid tumors, remain to be established. We show here in prostate cancer cells that increased NOXA, mediated by kinase inhibitor activation of an integrated stress response, drives the degradation of MCL1, and identify the mitochondria-associated ubiquitin ligase MARCH5 as the primary mediator of this NOXA-dependent MCL1 degradation. Therapies that enhance MARCH5-mediated MCL1 degradation markedly enhance apoptosis in response to a BH3 mimetic agent targeting BCLXL, which may provide for a broadly effective therapy in solid tumors. Conversely, increased MCL1 in response to MARCH5 loss does not sensitize to BH3 mimetic drugs targeting MCL1, but instead also sensitizes to BCLXL inhibition, revealing a codependence between MARCH5 and MCL1 that may also be exploited in tumors with MARCH5 genomic loss.
    Keywords:  cancer biology; human
    DOI:  https://doi.org/10.7554/eLife.54954
  22. Nat Commun. 2020 Jun 03. 11(1): 2794
    A SAM-I riboswitch with the ability to sense and respond to uncharged initiator tRNA.
    Dong-Jie Tang, Xinyu Du, Qiang Shi, Jian-Ling Zhang, Yuan-Ping He, Yan-Miao Chen, Zhenhua Ming, Dan Wang, Wan-Ying Zhong, Yu-Wei Liang, Jin-Yang Liu, Jian-Ming Huang, Yun-Shi Zhong, Shi-Qi An, Hongzhou Gu, Ji-Liang Tang.
      All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.
    DOI:  https://doi.org/10.1038/s41467-020-16417-z
  23. Proc Natl Acad Sci U S A. 2020 Jun 01. pii: 201917569. [Epub ahead of print]
    Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components.
    Jorge R Espinosa, Jerelle A Joseph, Ignacio Sanchez-Burgos, Adiran Garaizar, Daan Frenkel, Rosana Collepardo-Guevara.
      One of the key mechanisms used by cells to control the spatiotemporal organization of their many components is the formation and dissolution of biomolecular condensates through liquid-liquid phase separation (LLPS). Using a minimal coarse-grained model that allows us to simulate thousands of interacting multivalent proteins, we investigate the physical parameters dictating the stability and composition of multicomponent biomolecular condensates. We demonstrate that the molecular connectivity of the condensed-liquid network-i.e., the number of weak attractive protein-protein interactions per unit of volume-determines the stability (e.g., in temperature, pH, salt concentration) of multicomponent condensates, where stability is positively correlated with connectivity. While the connectivity of scaffolds (biomolecules essential for LLPS) dominates the phase landscape, introduction of clients (species recruited via scaffold-client interactions) fine-tunes it by transforming the scaffold-scaffold bond network. Whereas low-valency clients that compete for scaffold-scaffold binding sites decrease connectivity and stability, those that bind to alternate scaffold sites not required for LLPS or that have higher-than-scaffold valencies form additional scaffold-client-scaffold bridges increasing stability. Proteins that establish more connections (via increased valencies, promiscuous binding, and topologies that enable multivalent interactions) support the stability of and are enriched within multicomponent condensates. Importantly, proteins that increase the connectivity of multicomponent condensates have higher critical points as pure systems or, if pure LLPS is unfeasible, as binary scaffold-client mixtures. Hence, critical points of accessible systems (i.e., with just a few components) might serve as a unified thermodynamic parameter to predict the composition of multicomponent condensates.
    Keywords:  biomolecular condensates; cell compartmentalization; liquid–liquid phase separation; membraneless organelles
    DOI:  https://doi.org/10.1073/pnas.1917569117
  24. Cell Metab. 2020 Jun 02. pii: S1550-4131(20)30249-7. [Epub ahead of print]31(6): 1047-1049
    Break on Through: Golgi-Derived Vesicles Aid in Mitochondrial Fission.
    Megan L Rasmussen, Gabriella L Robertson, Vivian Gama.
      Mitochondrial fission is sustained through contact with several organelles, including the endoplasmic reticulum, lysosomes, and the actin cytoskeleton. Nagashima et al. (2020) now demonstrate that PI(4)P-containing Golgi-derived vesicles also modulate mitochondrial fission, driven by Arf1 and PI(4)KIIIβ activity, identifying a new organelle contact involved in maintaining mitochondrial homeostasis.
    DOI:  https://doi.org/10.1016/j.cmet.2020.05.010
  25. Nature. 2020 Jun 03.
    Structure of the ER membrane complex, a transmembrane-domain insertase.
    Lin Bai, Qinglong You, Xiang Feng, Amanda Kovach, Huilin Li.
      The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.
    DOI:  https://doi.org/10.1038/s41586-020-2389-3
  26. Hepatol Commun. 2020 Jun;4(6): 932-944
    Ritonavir and Lopinavir Suppress RCE1 and CAAX Rab Proteins Sensitizing the Liver to Organelle Stress and Injury.
    Atousa Khalatbari, Pratibha Mishra, Hui Han, Yuxin He, Michelle MacVeigh-Aloni, Cheng Ji.
      Organelle stress and Liver injuries often occur in human immunodeficiency virus (HIV) infected patients under anti-HIV therapies, yet few molecular off-targets of anti-HIV drugs have been identified in the liver. Here, we found through total RNA sequencing that the transcription of a host protease Ras converting CAAX endopeptidase 1 (RCE1) was altered in HepG2 cells treated with anti-HIV protease inhibitors, ritonavir and lopinavir. Levels of RCE1 protein were inhibited in HepG2 and primary mouse hepatocytes and in the liver of mice treated with the anti-HIV drugs, which were accompanied with inhibition of two potential substrates of RCE1, small GTP binding protein Rab13 and Rab18, which are with a common CAAX motif and known to regulate the ER-Golgi traffic or lipogenesis. Neither Rce1 transcription nor RCE1 protein level was inhibited by Brefeldin A, which is known to interfere with the ER-Golgi traffic causing Golgi stress. Knocking down Rce1 with RNA interference increased ritonavir and lopinavir-induced cell death as well as expression of Golgi stress response markers, TFE3, HSP47 and GCP60, in both primary mouse hepatocytes and mouse liver, and deteriorated alcohol-induced alanine aminotransferase (ALT) and fatty liver injury in mice. In addition, overexpressing Rab13 or Rab18 in primary human hepatocytes reduced partially the anti-HIV drugs and alcohol-induced Golgi fragmentation, Golgi stress response, and cell death injury. Conclusion: We identified a mechanism linking a host protease and its substrates, small guanosine triphosphate-binding proteins, to the anti-HIV drug-induced Golgi dysfunction, organelle stress response, and fatty liver injury.
    DOI:  https://doi.org/10.1002/hep4.1515
  27. Cell Death Differ. 2020 Jun 01.
    Olig2 SUMOylation protects against genotoxic damage response by antagonizing p53 gene targeting.
    Huiqing Liu, Weiji Weng, Rongjun Guo, Jie Zhou, Jun Xue, Shan Zhong, Jinke Cheng, Michael X Zhu, Si-Jian Pan, Yong Li.
      Posttranslational modifications of nuclear proteins, including transcription factors, nuclear receptors, and their coregulators, have attracted much attention in cancer research. Although phosphorylation of oligodendrocyte transcription factor 2 (Olig2) may contribute to the notorious resistance of gliomas to radiation and genotoxic drugs, the precise mechanisms remain elusive. We show here that in addition to phosphorylation, Olig2 is also conjugated by small ubiquitin-like modifier-1 (SUMO1) at three lysine residues K27, K76, and K112. SUMOylation is required for Olig2 to suppress p53-mediated cell cycle arrest and apoptosis induced by genotoxic damage, and to enhance resistance to temozolomide (TMZ) in glioma. Both SUMOylation and triple serine motif (TSM) phosphorylation of Olig2 are required for the antiapoptotic function. Olig2 SUMOylation enhances its genetic targeting ability, which in turn occludes p53 recruitment to Cdkn1a promoter for DNA-damage responses. Our work uncovers a SUMOylation-dependent regulatory mechanism of Olig2 in regulating cancer survival.
    DOI:  https://doi.org/10.1038/s41418-020-0569-1
  28. Bioessays. 2020 Jun 02. e1900256
    KCTD10 Biology: An Adaptor for the Ubiquitin E3 Complex Meets Multiple Substrates: Emerging Divergent Roles of the cullin-3/KCTD10 E3 Ubiquitin Ligase Complex in Various Cell Lines.
    Masashi Maekawa, Shigeki Higashiyama.
      Protein ubiquitination constitutes a post-translational modification mediated by ubiquitin ligases whereby ubiquitinated substrates are degraded through the proteasomal or lysosomal pathways, or acquire novel molecular functions according to their "ubiquitin codes." Dysfunction of the ubiquitination process in cells causes various diseases such as cancers along with neurodegenerative, auto-immune/inflammatory, and metabolic diseases. KCTD10 functions as a substrate recognition receptor for cullin-3 (CUL3), a scaffold protein in RING-type ubiquitin ligase complexes. Recently, studies by ourselves and others have identified new substrates that are ubiquitinated by the CUL3/KCTD10 ubiquitin ligase complex. Moreover, the type of polyubiquitination (e.g., K27-, K48-, or K63-chain) of various substrates (e.g., RhoB, CEP97, EIF3D, and TRIF) mediated by KCTD10 underlies its divergent roles in endothelial barrier formation, primary cilium formation, plasma membrane dynamics, cell proliferation, and immune response. Here, the physiological functions of KCTD10 are summarized and potential mechanisms are proposed.
    Keywords:  CEP97; EIF3D; KCTD10; RhoB; TRIF; cullin-3; ubiquitin E3 ligase
    DOI:  https://doi.org/10.1002/bies.201900256
  29. Molecules. 2020 May 28. pii: E2517. [Epub ahead of print]25(11):
    Development of Resistance to Endoplasmic Reticulum Stress-Inducing Agents in Mouse Leukemic L1210 Cells.
    Martin Cagala, Lucia Pavlikova, Mario Seres, Karolina Kadlecikova, Albert Breier, Zdena Sulova.
      Four new variants of L1210 cells resistant to endoplasmic reticulum (ER) stressors, tunicamycin (STun), thapsigargin (SThap), bortezomib (SBor), and MG-132 (SMG-132), were developed via an 18-month periodic cultivation in culture medium with a gradual increase in substance concentration. Multidrug resistance was generated for STun (to tunicamycin, bortezomib and MG-132), SThap (to tunicamycin, thapsigargin and MG-132), SBor (to bortezomib and MG-132), and SMG-132 (to bortezomib and MG-132). These cells were compared to the original L1210 cells and another two variants, which expressed P-gp due to induction with vincristine or transfection with the gene encoding P-gp, in terms of the following properties: sensitivity to either vincristine or the ER stressors listed above, proliferative activity, expression of resistance markers and proteins involved in the ER stress response, and proteasome activity. The resistance of the new cell variants to ER stressors was accompanied by a decreased proliferation rate and increased proteasome activity. The most consistent change in protein expression was the elevation of GRP78/BiP at the mRNA and protein levels in all resistant variants of L1210 cells. In conclusion, the mechanisms of resistance to these stressors have certain common features, but there are also specific differences.
    Keywords:  L1210 cells; MG-132; bortezomib; endoplasmic reticulum stress; multiple drug resistance; proteasomal activity; thapsigargin; tunicamycin; vincristine
    DOI:  https://doi.org/10.3390/molecules25112517
  30. J Biol Chem. 2020 06 02. pii: jbc.REV120.012669. [Epub ahead of print]
    Transcription factors activated through RIP (Regulated Intramembrane Proteolysis) and RAT (Regulated Alternative Translocation)).
    Jin Ye.
      Transmembrane proteins are membrane-anchored proteins whose topologies are important for their functions. These properties enable regulation of certain transmembrane proteins by regulated intramembrane proteolysis (RIP) and regulated alternative translocation (RAT). RIP enables a protein fragment of a transmembrane precursor to function at a new location, and RAT leads to an inverted topology of a transmembrane protein by altering the direction of its translocation across membranes during translation. RIP mediated by Site-1 protease (S1P) and Site-2 protease (S2P) are involved in proteolytic activation of membrane-bound transcription factors. In resting cells, these transcription factors remain in the endoplasmic reticulum (ER) as inactive transmembrane precursors. Upon stimulation by signals within the ER, they are translocated from the ER to the Golgi. There, they are cleaved first by S1P and then by S2P, liberating their N-terminal domains from membranes and enabling them to activate genes in the nucleus. This signaling pathway regulates lipid metabolism, unfolded protein responses, secretion of extracellular matrix proteins, and cell proliferation.  Remarkably, ceramide-induced RIP of cAMP response element-binding protein 3-like 1 (CREB3L1) also involves RAT. In resting cells, RIP of CREB3L1 is blocked by transmembrane 4 L6 family member 20 (TM4SF20). Ceramide inverts the orientation of newly synthesized TM4SF20 in membranes through RAT, converting TM4SF20 from an inhibitor to an activator of RIP of CREB3L1. Here, I review recent insights into RIP of membrane-bound transcription factors, focusing on CREB3L1 activation through both RIP and RAT, and discuss current open questions about these two signaling pathways.
    Keywords:  Golgi; RAT; RIP; Topology; ceramide; endoplasmic reticulum (ER); protein translocation; proteolysis; transcription factor; transmembrane domain; transport
    DOI:  https://doi.org/10.1074/jbc.REV120.012669
  31. Int J Mol Sci. 2020 May 28. pii: E3842. [Epub ahead of print]21(11):
    Valosin-Containing Protein, a Calcium-Associated ATPase Protein, in Endoplasmic Reticulum and Mitochondrial Function and Its Implications for Diseases.
    Xiaonan Sun, Hongyu Qiu.
      Endoplasmic reticulum (ER) and mitochondrion are the key organelles in mammal cells and play crucial roles in a variety of biological functions in both physiological and pathological conditions. Valosin-containing protein (VCP), a newly identified calcium-associated ATPase protein, has been found to be involved in both ER and mitochondrial function. Impairment of VCP, caused by structural mutations or alterations of expressions, contributes to the development of various diseases, through an integrating effect on ER, mitochondria and the ubiquitin-proteasome system, by interfering with protein degradation, subcellular translocation and calcium homeostasis. Thus, understanding the role and the molecular mechanisms of VCP in these organelles brings new insights to the pathogenesis of the associated diseases, and leads to the discovery of new therapeutic strategies. In this review, we summarized the progress of studies on VCP, in terms of its regulation of ER and mitochondrial function and its implications for the associated diseases, focusing on the cancers, heart disease, and neurodegenerative disorders.
    Keywords:  calcium homeostasis; disease; endoplasmic reticulum; mitochondria; valosin-containing protein
    DOI:  https://doi.org/10.3390/ijms21113842
  32. Biochim Biophys Acta Mol Cell Res. 2020 Jun 02. pii: S0167-4889(20)30123-3. [Epub ahead of print] 118765
    Src couples estrogen receptor to the anticipatory unfolded protein response and regulates cancer cell fate under stress.
    Liqun Yu, Lawrence Wang, Ji Eun Kim, Chengjian Mao, David J Shapiro.
      Accumulation of unfolded protein, or other stresses, activates the classical reactive unfolded protein response (UPR). In the recently characterized anticipatory UPR, receptor-bound estrogen, progesterone and other mitogenic hormones rapidly elicit phosphorylation of phospholipase C γ (PLCγ), activating the anticipatory UPR. How estrogen and progesterone activating their receptors couples to PLCγ phosphorylation and anticipatory UPR activation was unknown. We show that the oncogene c-Src is a rate-limiting regulator whose tyrosine kinase activity links estrogen and progesterone activating their receptors to anticipatory UPR activation. Supporting Src coupling estrogen and progesterone to anticipatory UPR activation, we identified extranuclear complexes of estrogen receptor α (ERα):Src:PLCγ and progesterone receptor:Src:PLCγ. Moreover, Src inhibition protected cancer cells against cell death. To probe Src's role, we used the preclinical ERα biomodulator, BHPI, which kills cancer cells by inducing lethal anticipatory UPR hyperactivation. Notably, Src inhibition blocked BHPI-mediated anticipatory UPR activation and the resulting rapid increase in intracellular calcium. After unbiased long-term selection for BHPI-resistant human breast cancer cells, 4/11 BHPI-resistant T47D clones, and nearly all MCF-7 clones, exhibited reduced levels of normally growth-stimulating Src. Notably, Src overexpression by virus transduction restored sensitivity to BHPI. Furthermore, in wild type cells, several-fold knockdown of Src, but not of ERα, strongly blocked BHPI-mediated UPR activation and subsequent HMGB1 release and necrotic cell death. Thus, Src plays a previously undescribed pivotal role in activation of the tumor-protective anticipatory UPR, thereby increasing the resilience of breast cancer cells. This is a new role for Src and the anticipatory UPR in breast cancer.
    Keywords:  Calcium; Cancer cell death; Dasatinib; Estrogen receptor; Src; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.bbamcr.2020.118765
  33. Sci Adv. 2020 Apr;6(16): eaaz7086
    O-GlcNAc transferase promotes influenza A virus-induced cytokine storm by targeting interferon regulatory factor-5.
    Qiming Wang, Peining Fang, Rui He, Mengqi Li, Haisheng Yu, Li Zhou, Yu Yi, Fubing Wang, Yuan Rong, Yi Zhang, Aidong Chen, Nanfang Peng, Yong Lin, Mengji Lu, Ying Zhu, Guoping Peng, Liqun Rao, Shi Liu.
      In this study, we demonstrated an essential function of the hexosamine biosynthesis pathway (HBP)-associated O-linked β-N-acetylglucosamine (O-GlcNAc) signaling in influenza A virus (IAV)-induced cytokine storm. O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, mediated IAV-induced cytokine production. Upon investigating the mechanisms driving this event, we determined that IAV induced OGT to bind to interferon regulatory factor-5 (IRF5), leading to O-GlcNAcylation of IRF5 on serine-430. O-GlcNAcylation of IRF5 is required for K63-linked ubiquitination of IRF5 and subsequent cytokine production. Analysis of clinical samples revealed that IRF5 is O-GlcNAcylated, and higher levels of proinflammatory cytokines correlated with higher levels of blood glucose in IAV-infected patients. We identified a molecular mechanism by which HBP-mediated O-GlcNAcylation regulates IRF5 function during IAV infection, highlighting the importance of glucose metabolism in IAV-induced cytokine storm.
    DOI:  https://doi.org/10.1126/sciadv.aaz7086
  34. PLoS One. 2020 ;15(6): e0234147
    Enhanced phosphorylation of PERK in primary cultured neurons as an autonomous neuronal response to prion infection.
    Misaki Tanaka, Takeshi Yamasaki, Rie Hasebe, Akio Suzuki, Motohiro Horiuchi.
      Conversion of cellular prion protein (PrPC) into the pathogenic isoform of prion protein (PrPSc) in neurons is one of the key pathophysiological events in prion diseases. However, the molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrPSc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrPSc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2α) pathway, demonstrated a positive correlation between the number of PrPSc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2α pathway. These results suggest that PrPSc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.
    DOI:  https://doi.org/10.1371/journal.pone.0234147
  35. J Biol Chem. 2020 Jun 04. pii: jbc.REV120.011473. [Epub ahead of print]
    Role of the lipid bilayer in outer membrane protein folding in Gram negative bacteria.
    Jim E Horne, David J Brockwell, Sheena E Radford.
      β-barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonisation and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP folding landscape and discuss the factors that guide folding in vitro and in vivo. We particularly focus on the composition, architecture and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo. Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. Since OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance, to bring down the powerful, OMP-supported barrier against antibiotics, we must first understand how bacterial cells build it.
    Keywords:  BAM complex; lipid; membrane bilayer; membrane protein; membrane protein folding; outer membrane; protein folding; protein-lipid interactions
    DOI:  https://doi.org/10.1074/jbc.REV120.011473
  36. mBio. 2020 Jun 02. pii: e00979-20. [Epub ahead of print]11(3):
    NOD1/NOD2 and RIP2 Regulate Endoplasmic Reticulum Stress-Induced Inflammation during Chlamydia Infection.
    Oanh H Pham, Bokyung Lee, Jasmine Labuda, A Marijke Keestra-Gounder, Mariana X Byndloss, Renée M Tsolis, Stephen J McSorley.
      The inflammatory response to Chlamydia infection is likely to be multifactorial and involve a variety of ligand-dependent and -independent recognition pathways. We previously reported the presence of NOD1/NOD2-dependent endoplasmic reticulum (ER) stress-induced inflammation during Chlamydia muridarum infection in vitro, but the relevance of this finding to an in vivo context is unclear. Here, we examined the ER stress response to in vivo Chlamydia infection. The induction of interleukin 6 (IL-6) production after systemic Chlamydia infection correlated with expression of ER stress response genes. Furthermore, when tauroursodeoxycholate (TUDCA) was used to inhibit the ER stress response, an increased bacterial burden was detected, suggesting that ER stress-driven inflammation can contribute to systemic bacterial clearance. Mice lacking both NOD1 and NOD2 or RIP2 exhibited slightly higher systemic bacterial burdens after infection with Chlamydia Overall, these data suggest a model where RIP2 and NOD1/NOD2 proteins link ER stress responses with the induction of Chlamydia-specific inflammatory responses.IMPORTANCE Understanding the initiation of the inflammatory response during Chlamydia infection is of public health importance given the impact of this disease on young women in the United States. Many young women are chronically infected with Chlamydia but are asymptomatic and therefore do not seek treatment, leaving them at risk of long-term reproductive harm due to inflammation in response to infection. Our manuscript explores the role of the endoplasmic reticulum stress response pathway initiated by an innate receptor in the development of this inflammation.
    Keywords:  Chlamydia ; ER stress; NOD; inflammation; innate immunity
    DOI:  https://doi.org/10.1128/mBio.00979-20
  37. J Biol Chem. 2020 Jun 01. pii: jbc.RA120.013454. [Epub ahead of print]
    A-kinase-anchoring protein 1 (dAKAP1)-based signaling complexes coordinate local protein synthesis at the mitochondrial surface.
    Laura Gabrovsek, Kerrie B Collins, Stacey Aggarwal, Lauren M Saunders, Ho-Tak Lau, Danny Suh, Yasemin Sancak, Cole Trapnell, Shao-En Ong, F Donelson Smith, John D Scott.
      Compartmentalization of macromolecules is a ubiquitous molecular mechanism that drives numerous cellular functions. Appropriate organization of enzymes in space and time enables the precise transmission and integration of intracellular signals. Molecular scaffolds constrain signaling enzymes to influence the regional modulation of these physiological processes.  Mitochondrial targeting of protein kinases and protein phosphatases provides a means to locally control the phosphorylation status and action of proteins on the surface of this organelle. Dual-specificity A-kinase anchoring protein 1 (dAKAP1) is a multivalent binding protein that targets protein kinase A (PKA), RNAs and other signaling enzymes to the outer mitochondrial membrane. Many AKAPs recruit a diverse set of binding partners that coordinate a broad range of cellular process. Here, results of mass spectrometry and biochemical analyses reveal that dAKAP1 anchors additional components including the ribonucleoprotein granule components La-related protein 4 (LARP4) and polyadenylate-binding protein 1 (PABPC1). Local translation of mRNAs at organelles is a means to spatially control the synthesis of proteins. RNA-Seq data demonstrate that dAKAP1 binds mRNAs encoding proteins required for mitochondrial metabolism, including succinate dehydrogenase. Functional studies suggest that loss of dAKAP1-RNA interactions reduces mitochondrial electron transport chain activity. Hence, dAKAP1 plays a previously unappreciated role as a molecular interface between second messenger signaling and local protein synthesis machinery.
    Keywords:  A-kinase anchoring protein (AKAP); RNA-protein interaction; local signaling complexes; mitochondria; protein kinase; protein kinase A (PKA)
    DOI:  https://doi.org/10.1074/jbc.RA120.013454
  38. J Biochem. 2020 Jun 02. pii: mvaa061. [Epub ahead of print]
    The Coupling of Translational Control and Stress Responses.
    Ryan Houston, Shiori Sekine, Yusuke Sekine.
      The translation of messenger RNA (mRNA) into protein is a multistep process by which genetic information transcribed into an mRNA is decoded to produce a specific polypeptide chain of amino acids. Ribosomes play a central role in translation by coordinately working with various translation regulatory factors and aminoacyl-transfer RNAs (tRNAs). A variety of stresses attenuate the ribosomal synthesis in the nucleolus as well as the translation rate in the cytosol. To efficiently reallocate cellular energy and resources, mammalian cells are endowed with mechanisms that directly link the suppression of translation-related processes to the activation of stress adaptation programs. This review focuses on the integrated stress response (ISR) and the nucleolar stress response (NSR) both of which are activated by various stressors and selectively upregulate stress-responsive transcription factors. Emerging findings have delineated the detailed molecular mechanisms of the ISR and NSR and expanded their physiological and pathological significances.
    Keywords:  integrated stress response; nucleolar stress response; protein synthesis; ribosome biosynthesis; translation initiation
    DOI:  https://doi.org/10.1093/jb/mvaa061
  39. EMBO J. 2020 Jun 02. e103838
    Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.
    Joffrey Pelletier, Ferran Riaño-Canalias, Eugènia Almacellas, Caroline Mauvezin, Sara Samino, Sonia Feu, Sandra Menoyo, Ana Domostegui, Marta Garcia-Cajide, Ramon Salazar, Constanza Cortés, Ricard Marcos, Albert Tauler, Oscar Yanes, Neus Agell, Sara C Kozma, Antonio Gentilella, George Thomas.
      Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability.
    Keywords:   IRBC ; IMPDH; nucleotides; p21; p53
    DOI:  https://doi.org/10.15252/embj.2019103838
  40. J Microsc. 2020 Jun 04.
    Light microscopy of the endoplasmic reticulum-membrane contact sites in plants.
    J Zang, T Zhang, P J Hussey, P Wang.
      The existence of membrane contact sites (MCS) has been reported in different systems in the past decade, and their importance has been recognized by the cell biology community. Amongst all endomembrane structures, the endoplasmic reticulum (ER) plays vital roles in organizing the organelle interaction network with the plasma membrane (PM), Golgi bodies, mitochondria, plastids, endosomes and autophagosomes. A number of methods have been used to study the establishment and functions of these interactions, among them, light microscopy appears to be one of the most effective approaches. Here, we present an overview of the discovery of ER-PM contact sites, and highlight the latest developments in light microscopical-based techniques that can be used for their study. This article is protected by copyright. All rights reserved.
    Keywords:  ER-PM contact sites; Endoplasmic Reticulum; Light Microscopy; Membrane Contact Sites; Plants
    DOI:  https://doi.org/10.1111/jmi.12934
  41. Cell Mol Neurobiol. 2020 Jun 05.
    The Regulatory Role of Reticulons in Neurodegeneration: Insights Underpinning Therapeutic Potential for Neurodegenerative Diseases.
    Lilesh Kumar Pradhan, Saroj Kumar Das.
      In the last few decades, cytoplasmic organellar dysfunction, such as that of the endoplasmic reticulum (ER), has created a new area of research interest towards the development of serious health maladies including neurodegenerative diseases. In this context, the extensively dispersed family of ER-localized proteins, i.e. reticulons (RTNs), is gaining interest because of its regulative control over neural regeneration. As most neurodegenerative diseases are pathologically manifested with the accretion of misfolded proteins with subsequent induction of ER stress, the regulatory role of RTNs in neural dysfunction cannot be ignored. With the limited information available in the literature, delineation of the functional connection between rising consequences of neurodegenerative diseases and RTNs need to be elucidated. In this review, we provide a broad overview on the recently revealed regulatory roles of reticulons in the pathophysiology of several health maladies, with special emphasis on neurodegeneration. Additionally, we have also recapitulated the decisive role of RTN4 in neurite regeneration and highlighted how neurodegeneration and proteinopathies are mechanistically linked with each other through specific RTN paralogues. With the recent findings advocating zebrafish Rtn4b (a mammalian Nogo-A homologue) downregulation following central nervous system (CNS) lesion, RTNs provides new insight into the CNS regeneration. However, there are controversies with respect to the role of Rtn4b in zebrafish CNS regeneration. Given these controversies, the connection between the unique regenerative capabilities of zebrafish CNS by distinct compensatory mechanisms and Rtn4b signalling pathway could shed light on the development of new therapeutic strategies against serious neurodegenerative diseases.
    Keywords:  CNS regeneration; Endoplasmic reticulum; Neurodegeneration; Neurodegenerative diseases; Nogo-A; Reticulons; Rtn4b
    DOI:  https://doi.org/10.1007/s10571-020-00893-4
  42. Mol Cell. 2020 Jun 04. pii: S1097-2765(20)30303-8. [Epub ahead of print]78(5): 811-813
    ER-phagy: Eating the Factory.
    Maurizio Molinari.
      Liang et al. (2020) reports on a genome-wide screen that reveals new aspects of starvation-induced degradation of the endoplasmic reticulum.
    DOI:  https://doi.org/10.1016/j.molcel.2020.05.002
  43. Autophagy. 2020 Jun 05.
    Autophagy coupled to translation is required for long-term memory.
    Kiran Pandey, Xiao-Wen Yu, Adam Steinmetz, Cristina M Alberini.
      An increase in protein synthesis following learning is a fundamental and evolutionarily conserved mechanism of long-term memory. To maintain homeostasis, this protein synthesis must be counterbalanced by mechanisms such as protein degradation. Recent studies reported that macroautophagy/autophagy, a major protein degradation mechanism, is required for long-term memory formation. However, how learning regulates autophagy and recruits it into long-term memory formation remains to be established. Here, we show that inhibitory avoidance in rats significantly increases the levels of autophagy and lysosomal degradation proteins, including BECN1/beclin 1, LC3-II, SQSTM1/p62 and LAMP1, as well as autophagic flux in the hippocampus. Moreover, pharmacological inhibition or targeted molecular disruption of the learning-induced autophagy impairs long-term memory, leaving short-term memory intact. The increase in autophagy proteins results from active translation of their mRNA and not from changes in their total mRNA levels. Additionally, the induction of autophagy requires the immediate early gene Arc/Arg3.1. Finally, in contrast to classical regulation of autophagy in other systems, we found that the increase in autophagy upon learning is dispensable for the increase in protein synthesis. We conclude that coupling between learning-induced translation and autophagy, rather than translation per se, is an essential mechanism of long-term memory.
    Keywords:  Arc/Arg3.1; autophagic flux; autophagy; learning; memory; translating ribosome affinity purification; translation
    DOI:  https://doi.org/10.1080/15548627.2020.1775393
  44. J Toxicol Sci. 2020 ;45(6): 305-317
    Endoplasmic reticulum stress mediated apoptosis via JNK in MWCNT-exposed in vitro systems: size, surface functionalization and cell type specificity.
    Nivedita Chatterjee, Jinhee Choi.
      The aim of the present study was to evaluate the underlying mechanism of multi-walled carbon nanotubes (MWCNT) induced cellular response and their potential cross-talk, specifically, between endoplasmic reticulum (ER) stress, MAPK activation and apoptosis and how these nano-bio interactions depend on the physico-chemical properties of MWCNT. For this purpose, human bronchial epithelial (Beas2B) and human hepatoma (HepG2) cell lines, were exposed to five kinds of MWCNTs which differ in functionalization and aspect ratios. Tissue-specific sensitivity was evident for calcium homeostasis, ER-stress response, MAPK activation and apoptosis, which further depended on surface functionalization as well as aspect ratios of MWCNT. By applying specific pharmaceutical inhibitors, relevant biomarkers gene and proteins expressions, we found that possibly MWCNT induce activation of IRE1α-XPB1 pathway-mediated ER-stress response, which in turn trigger apoptosis through JNK activation in both type of cells but with variable intensity. The information presented here would have relevance in better understanding of MWCNT toxicity and their safer applications.
    Keywords:  Apoptosis; Calcium homeostasis; Endoplasmic reticulum stress; In vitro cell type specificity; MAPK activation; MWCNTs
    DOI:  https://doi.org/10.2131/jts.45.305
  45. ACS Infect Dis. 2020 Jun 05.
    HSF1 Activation Can Restrict HIV Replication.
    Emmanuel E Nekongo, Anna I Ponomarenko, Mahender B Dewal, Vincent L Butty, Edward P Browne, Matthew D Shoulders.
      Host protein folding stress responses can play important roles in RNA virus replication and evolution. Prior work suggested a complicated interplay between the cytosolic proteostasis stress response, controlled by the transcriptional master regulator heat shock factor 1 (HSF1), and human immunodeficiency virus-1 (HIV-1). We sought to uncouple HSF1 transcription factor activity from cytotoxic proteostasis stress and thereby better elucidate the proposed role(s) of HSF1 in the HIV-1 lifecycle. To achieve this objective, we used chemical genetic, stress-independent control of HSF1 activity to establish whether and how HSF1 influences HIV-1 replication. Stress-independent HSF1 induction decreased both the total quantity and infectivity of HIV-1 virions. Moreover, HIV-1 was unable to escape HSF1-mediated restriction over the course of several serial passages. These results clarify the interplay between the host's heat shock response and HIV-1 infection, and promote continued consideration of chaperones as potential antiviral therapeutic targets.
    DOI:  https://doi.org/10.1021/acsinfecdis.0c00166
  46. EMBO Mol Med. 2020 Jun 05. e11861
    Proteasome subunit PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress.
    Ariane Kröll-Hermi, Frédéric Ebstein, Corinne Stoetzel, Véronique Geoffroy, Elise Schaefer, Sophie Scheidecker, Séverine Bär, Masanari Takamiya, Koichi Kawakami, Barbara A Zieba, Fouzia Studer, Valerie Pelletier, Carine Eyermann, Claude Speeg-Schatz, Vincent Laugel, Dan Lipsker, Florian Sandron, Steven McGinn, Anne Boland, Jean-François Deleuze, Lauriane Kuhn, Johana Chicher, Philippe Hammann, Sylvie Friant, Christelle Etard, Elke Krüger, Jean Muller, Uwe Strähle, Hélène Dollfus.
      The ubiquitin-proteasome system degrades ubiquitin-modified proteins to maintain protein homeostasis and to control signalling. Whole-genome sequencing of patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient's fibroblasts was however unaffected. Nevertheless, patient's cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient's fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient's cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development.
    Keywords:   PSMC3 ; cataract; deafness; neurosensory disease; proteasome
    DOI:  https://doi.org/10.15252/emmm.201911861
  47. J Clin Invest. 2020 Jun 01. pii: 132876. [Epub ahead of print]130(6): 3253-3269
    Parkin ubiquitinates phosphoglycerate dehydrogenase to suppress serine synthesis and tumor progression.
    Juan Liu, Cen Zhang, Hao Wu, Xiao-Xin Sun, Yanchen Li, Shan Huang, Xuetian Yue, Shou-En Lu, Zhiyuan Shen, Xiaoyang Su, Eileen White, Bruce G Haffty, Wenwei Hu, Zhaohui Feng.
      Phosphoglycerate dehydrogenase (PHGDH), the first rate-limiting enzyme of serine synthesis, is frequently overexpressed in human cancer. PHGDH overexpression activates serine synthesis to promote cancer progression. Currently, PHGDH regulation in normal cells and cancer is not well understood. Parkin, an E3 ubiquitin ligase involved in Parkinson's disease, is a tumor suppressor. Parkin expression is frequently downregulated in many types of cancer, and its tumor-suppressive mechanism is poorly defined. Here, we show that PHGDH is a substrate for Parkin-mediated ubiquitination and degradation. Parkin interacted with PHGDH and ubiquitinated PHGDH at lysine 330, leading to PHGDH degradation to suppress serine synthesis. Parkin deficiency in cancer cells stabilized PHGDH and activated serine synthesis to promote cell proliferation and tumorigenesis, which was largely abolished by targeting PHGDH with RNA interference, CRISPR/Cas9 KO, or small-molecule PHGDH inhibitors. Furthermore, Parkin expression was inversely correlated with PHGDH expression in human breast cancer and lung cancer. Our results revealed PHGDH ubiquitination by Parkin as a crucial mechanism for PHGDH regulation that contributes to the tumor-suppressive function of Parkin and identified Parkin downregulation as a critical mechanism underlying PHGDH overexpression in cancer.
    Keywords:  Metabolism; Oncology; Tumor suppressors; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI132876
  48. J Cell Biol. 2020 Aug 03. pii: e201903127. [Epub ahead of print]219(8):
    ERdj8 governs the size of autophagosomes during the formation process.
    Yo-Hei Yamamoto, Ayano Kasai, Hiroko Omori, Tomoe Takino, Munechika Sugihara, Tetsuo Umemoto, Maho Hamasaki, Tomohisa Hatta, Tohru Natsume, Richard I Morimoto, Ritsuko Arai, Satoshi Waguri, Miyuki Sato, Ken Sato, Shoshana Bar-Nun, Tamotsu Yoshimori, Takeshi Noda, Kazuhiro Nagata.
      In macroautophagy, membrane structures called autophagosomes engulf substrates and deliver them for lysosomal degradation. Autophagosomes enwrap a variety of targets with diverse sizes, from portions of cytosol to larger organelles. However, the mechanism by which autophagosome size is controlled remains elusive. We characterized a novel ER membrane protein, ERdj8, in mammalian cells. ERdj8 localizes to a meshwork-like ER subdomain along with phosphatidylinositol synthase (PIS) and autophagy-related (Atg) proteins. ERdj8 overexpression extended the size of the autophagosome through its DnaJ and TRX domains. ERdj8 ablation resulted in a defect in engulfing larger targets. C. elegans, in which the ERdj8 orthologue dnj-8 was knocked down, could perform autophagy on smaller mitochondria derived from the paternal lineage but not the somatic mitochondria. Thus, ERdj8 may play a critical role in autophagosome formation by providing the capacity to target substrates of diverse sizes for degradation.
    DOI:  https://doi.org/10.1083/jcb.201903127
  49. Neurochem Res. 2020 Jun 04.
    A Critical Role for ISGylation, Ubiquitination and, SUMOylation in Brain Damage: Implications for Neuroprotection.
    Venkata Prasuja Nakka, Abdul Qadeer Mohammed.
      Post-translational modification (PTMs) of proteins by ubiquitin and ubiquitin-like modifiers such as interferon-stimulated gene 15 (ISG15) and small ubiquitin-related modifier (SUMO) play a critical role in the regulation of brain pathophysiology. Protein ISGylation is a covalent attachment of ISG15 to its target proteins, which is a unique PTM among other ubiquitin-like modifiers. Although, ISG15 shares sequence homology to ubiquitin, yet the functional significance of protein ISGylation is distinct from ubiquitination and SUMOylation. Further, ISG15 highly conserved among vertebrate species, unlike the other ubiquitin-like modifiers. ISGylation modulates various intracellular mechanisms such as Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway, autophagy, DNA repair, etc., indicating its biological significance. ISGylation emerged as one of the important mechanisms in the regulation of various neurological disorders including stroke, traumatic brain injury (TBI), basal ganglia calcification, and ataxia-telangiectasia. It appears that protein ISGylation is an endogenous neuroprotective mechanism. This review discusses the role of ISGylation in various brain pathologies with a particular emphasis on cerebral ischemia/stroke and on structural similarities between ISG15 and ubiquitin. Further, recent advancements on the role of ubiquitination and SUMOylation with relevance to ISGylation will also be discussed. The overall goal is to provide better insights on the mechanistic link between ISGylation and other ubiquitin-like modifiers, which may be helpful to establish novel therapeutic strategies for neuroprotection.
    Keywords:  Brain damage; ISG15 conjugates; ISGylation; Post-translational modification; SUMOylation; Stroke; Ubiquitination
    DOI:  https://doi.org/10.1007/s11064-020-03066-3
  50. Nat Commun. 2020 Jun 05. 11(1): 2846
    Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~ubiquitin conformation.
    Emma Branigan, J Carlos Penedo, Ronald T Hay.
      Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.
    DOI:  https://doi.org/10.1038/s41467-020-16666-y
  51. Biochem Biophys Res Commun. 2020 Jun 01. pii: S0006-291X(20)30975-X. [Epub ahead of print]
    E3 ubiquitin ligase ATL61 acts as a positive regulator in abscisic acid mediated drought response in Arabidopsis.
    Ruirui Yang, Tao Wang, Wensen Shi, Siyu Li, Zhibin Liu, Jianmei Wang, Yi Yang.
      Abscisic acid (ABA) is an important plant hormone that mediates abiotic stresses in plant growth and development. A number of E3 ligases have been reported to be involved in ABA signaling pathway. In this study, we identified a C3H2C3 RING-type E3 ligase, Arabidopsis thaliana Tόxicos en Levadura 61 (ATL61), which regulated drought stress in planta. Enzyme assay in vitro demonstrated that ATL61 had E3 ubiquitin ligase activity, while point mutation of ATL61H109A, H122A (mATL61) abolished its E3 ubiquitin ligase activity. ATL61 overexpression plants exhibited ABA hypersensitivity and were more tolerant to drought, while the atl61 mutant plants were insensitive to ABA. Moreover, mATL61 overexpression lines exhibited similar ABA-related phenotypes with wild type (WT) plants. The transcript abundances of ABA-mediated drought stress-related genes RD20 and RD22 were higher in ATL61 overexpression plants than those in WT, atl61, and mATL61 plants. Our results indicated that ATL61 acted as a positive regulator in the ABA-mediated drought stress response.
    Keywords:  ATL61; Abscisic acid; Drought; E3 ligase
    DOI:  https://doi.org/10.1016/j.bbrc.2020.05.067
  52. Aging (Albany NY). 2020 Jun 03. 12
    UBE2T promotes glioblastoma invasion and migration via stabilizing GRP78 and regulating EMT.
    Peng Huang, Yuduo Guo, Zitong Zhao, Weihai Ning, Haoran Wang, Chunyu Gu, Mingshan Zhang, Yanming Qu, Hongwei Zhang, Yongmei Song.
      Glioblastoma (GBM) generally has a dismal prognosis, and it is associated with a poor quality of life as the disease progresses. However, the development of effective therapies for GBM has been deficient. Ubiquitin-conjugating enzyme E2T (UBE2T) is a member of the E2 family in the ubiquitin-proteasome pathway and a vital regulator of tumour progression, but its role in GBM is unclear. In this study, we aimed to clarify the role of UBE2T in GBM. Bioinformatics analysis identified UBE2T as an independent risk factor for gliomas. Immunohistochemistry was used to measure UBE2T expression in GBM and normal tissue samples obtained from patients with GBM. The effects of UBE2T on GBM cell invasion and migration were analysed using the Transwell assay. BALB/c nude mice were used for the in vivo assays. Immunoblotting and immunoprecipitation were performed to determine the molecular mechanisms. UBE2T was highly expressed in GBM tissues, and its expression was linked to a poor prognosis. In vitro, depletion of UBE2T significantly suppressed cell invasion and migration. Moreover, UBE2T depletion suppressed the growth of GBM subcutaneous tumours in vivo. Further experiments revealed that UBE2T suppressed invasion and migration by regulating epithelial- mesenchymal transition (EMT) via stabilising GRP78 in GBM cells. We uncovered a novel UBE2T/GRP78/EMT regulatory axis that modulates the malignant progression and recurrence of GBM, indicating that the axis might be a valuable therapeutic target.
    Keywords:  Endoplasmic Reticulum Lumenal Ca(2+)-Binding Protein Grp78; Ubiquitin-conjugating enzyme E2T; epithelial-mesenchymal transition; glioblastoma; therapeutic target
    DOI:  https://doi.org/10.18632/aging.103239
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