bims-proteo 2020-07-26 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2020–07–26
thirty-two papers selected by
Eric Chevet, INSERM

Vps13 is required for the packaging of the ER into autophagosomes during ER-phagy.
UAE1 inhibition mediates the unfolded protein response, DNA damage and caspase-dependent cell death in pancreatic cancer.
Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies.
CALCOCO1 is a soluble reticulophagy receptor.
Role of the O-GlcNAc modification on insulin resistance and endoplasmic reticulum stress in 3T3-L1 cells.
Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.
PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.
STEEP mediates STING ER exit and activation of signaling.
TMEM147 interacts with Lamin B Receptor, regulates its localization and levels, and affects cholesterol homeostasis.
The UPR in Neurodegenerative Disease: Not Just an Inside Job.
The ATF3 Transcription Factor Is a Short-Lived Substrate of the Arg/N-Degron Pathway.
Proteome-wide identification of HSP70/HSC70 chaperone clients in human cells.
Regulation of Histone Ubiquitination in Response to DNA Double Strand Breaks.
The role of host eIF2α in viral infection.
PtdIns4P restriction by hydrolase SAC1 decides specific fusion of autophagosomes with lysosomes.
Praja1 RING-finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP-43 aggregate formation.
Inhibition of the integrated stress response by viral proteins that block p-eIF2-eIF2B association.
Gcn2 eIF2α kinase mediates combinatorial translational regulation through nucleotide motifs and uORFs in target mRNAs.
N-linked glycan sites on the influenza NA head domain are required for efficient IAV incorporation and replication.
VIP conditions human endometrial receptivity by privileging endoplasmic reticulum stress through ATF6α pathway.
Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing.
Evidence for an involvement of the ubiquitin-like modifier ISG15 in MHC class I antigen presentation.
The COP9 Signalosome: A Multi-DUB Complex.
Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells.
Grouper ubiquitin-specific protease 14 promotes iridovirus replication through negatively regulating interferon response.
NRF2 activation promotes the recurrence of dormant tumour cells through regulation of redox and nucleotide metabolism.
Endoplasmic reticulum stress controls iron metabolism through TMPRSS6 repression and hepcidin mRNA stabilization by RNA-binding protein HuR.
Mitogen- and Stress-Activated Protein Kinase 1 Mediates Alcohol-Upregulated Transcription of Brf1 and tRNA Genes to Cause Phenotypic Alteration.
Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane-Proximal Regions of VAMP2 and SNAP25.
A nuclear role for Atg8-family proteins.
SUMO E3 ligase PIAS1 is a potential biomarker indicating stress susceptibility.
BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes.

Proc Natl Acad Sci U S A. 2020 Jul 20. pii: 202008923. [Epub ahead of print]

Vps13 is required for the packaging of the ER into autophagosomes during ER-phagy.

Shuliang Chen, Muriel Mari, Smriti Parashar, Dongmei Liu, Yixian Cui, Fulvio Reggiori, Peter J Novick, Susan Ferro-Novick.

  Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.

Keywords:  ER-phagy; Vps13; autophagy; contact site; lipid transporter

DOI:  https://doi.org/10.1073/pnas.2008923117
Transl Oncol. 2020 Jul 17. pii: S1936-5233(20)30326-0. [Epub ahead of print]13(11): 100834

UAE1 inhibition mediates the unfolded protein response, DNA damage and caspase-dependent cell death in pancreatic cancer.

Yajing Liu, Sahezeel Awadia, Amy Delaney, Merna Sitto, Carl Engelke, Heli Patel, Andrew Calcaterra, Sylvia Zelenka-Wang, Hojin Lee, Joseph Contessa, Nouri Neamati, Mats Ljungman, Theodore S Lawrence, Meredith A Morgan, Alnawaz Rehemtulla.

  The Unfolded Protein Response (UPR) plays a key role in the adaptive response to loss of protein homeostasis within the endoplasmic reticulum (ER). The UPR has an adaptive function in protein homeostasis, however, sustained activation of the UPR due to hypoxia, nutrient deprivation, and increased demand for protein synthesis, alters the UPR program such that additional perturbation of ER homeostasis activates a pro-apoptotic program. Since ubiquitination followed by proteasomal degradation of misfolded proteins within the ER is a central mechanism for restoration of ER homeostasis, inhibitors of this pathway have proven to be valuable anti-cancer therapeutics. Ubiquitin activating enzyme 1(UAE1), activates ubiquitin for transfer to target proteins for proteasomal degradation in conjunction with E2 and E3 enzymes. Inhibition of UAE1 activity in response to TAK-243, leads to an accumulation of misfolded proteins within the ER, thereby aggravating ER stress, leading to DNA damage and arrest of cells in the G2/M phase of the cell cycle. Persistent drug treatment mediates a robust induction of apoptosis following a transient cell cycle arrest. These biological effects of TAK-243 were recapitulated in mouse models of PDAC demonstrating antitumor activity at a dose and schedule that did not exhibit obvious normal tissue toxicity. In vitro as well as studies in mouse models failed to show enhanced efficacy when TAK-243 was combined with ionizing radiation or gemcitabine, providing an impetus for future studies to identify agents that synergize with this class of agents for improved tumor control in PDAC. SIGNIFICANCE: The UAE1 inhibitor TAK-243, mediates activation of the unfolded protein response, accumulation of DNA breaks and apoptosis, providing a rationale for the use as a safe and efficacious anti-cancer therapeutic for PDAC.

Keywords:  ER stress; Pancreatic cancer; UAE1 Inhibitor

DOI:  https://doi.org/10.1016/j.tranon.2020.100834
bioRxiv. 2020 Jul 14. pii: 2020.07.13.201517. [Epub ahead of print]

Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies.

Jonathan P Davies, Katherine M Almasy, Eli F McDonald, Lars Plate.

Human coronaviruses (hCoV) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite high sequence similarity between SARS-CoV-1 and -2, each strain has distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV non-structural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43 - an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologs from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca 2+ signaling and mitochondria biogenesis. We also identifiy nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N -linked glycosylation machinery, unfolded protein response (UPR) associated proteins, and anti-viral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondrial-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed anti-viral therapeutics.

DOI: https://doi.org/10.1101/2020.07.13.201517
Autophagy. 2020 Jul 20.

CALCOCO1 is a soluble reticulophagy receptor.

Thaddaeus Mutugi Nthiga, Birendra Kumar Shrestha, Trond Lamark, Terje Johansen.

  The endoplasmic reticulum (ER) is the largest membrane-bound organelle in eukaryotic cells and plays critical roles in diverse processes in metabolism, signaling and intracellular organization. In response to stress stimuli such as nutrient deprivation, accumulation of misfolded proteins or exposure to chemicals, the ER increases in size through upregulated synthesis of its components to counteract the stress. To restore physiological size, the excess ER components are continuously dismantled and degraded by reticulophagy, a form of autophagy that targets, via adaptor molecules called reticulophagy receptors, specific ER portions to the lysosome for degradation. Previous studies have identified several ER resident proteins as reticulophagy receptors. In a recent study, we identified CALCOCO1 as a soluble reticulophagy receptor for the degradation of tubular ER in response to proteotoxic and starvation-induced stress. On the ER membrane, CALCOCO1 interacts with VAPA and VAPB via a FFAT-like motif and recruits autophagy machinery by binding directly to Atg8-family proteins via LIR and UDS interacting region (UIR) motifs acting co-dependently. Depletion of CALCOCO1 in cultured cells led to an impaired ER degradation during stress.

Keywords:  Autophagy receptor; CALCOCO1; ER-phagy; FFAT motif; VAPA

DOI:  https://doi.org/10.1080/15548627.2020.1797289
Mol Biol Rep. 2020 Jul 20.

Role of the O-GlcNAc modification on insulin resistance and endoplasmic reticulum stress in 3T3-L1 cells.

Benan Pelin Sermikli, Gulizar Aydogdu, Erkan Yilmaz.

  O-linked N-acetyl-glucosamine (O-GlcNAc) is a post-translational protein modification that regulates cell signaling and involves in several pathological conditions. O-GlcNAc transferase (OGT) catalyzes the attachment, while O-GlcNAcase (OGA) splits the GlcNAc molecules from the serine or threonine residues of the nuclear and cellular proteins. The hexosamine biosynthesis pathway (HBP) is a small branch of glycolysis that provides a substrate for the OGT and serves as a nutrient sensor. In this study, we investigated the impact of external O-GlcNAc modification stimulus on the insulin signal transduction, unfolded protein response, and HBP in 3T3-L1 cells. First, we treated cells with glucosamine and PUGNAc to stimulate the O-GlcNAcylation of total proteins. Also, we treated cells with tunicamycin as a positive internal control, which is a widely-used endoplasmic reticulum stressor. We used two in vitro models to understand the impact of the cellular state of insulin sensibility on this hypothesis. So, we employed insulin-sensitive preadipocytes and insulin-resistant adipocytes to answer these questions. Secondly, the OGT-silencing achieved in the insulin-resistant preadipocyte model by using the short-hairpin RNA (shRNA) interference method. Thereafter, the cells treated with the above-mentioned compounds to understand the role of the diminished O-GlcNAc protein modification on the insulin signal transduction, unfolded protein response, and HBP. We found that elevated O-GlcNAcylation of the total proteins displayed a definite correlation in insulin resistance and endoplasmic reticulum stress. Furthermore, we identified that the degree of this correlation depends on the cellular state of insulin sensitivity. Moreover, reduced O-GlcNAcylation of the total proteins by the shRNA-mediated silencing of the OGT gene, which is the only gene to modify proteins with the O-GlcNAc molecule, reversed the insulin resistance and endoplasmic reticulum stress phenotype, even with the externally stimulated O-GlcNAc modification conditions. In conclusion, our results suggest that OGT regulates insulin receptor signaling and unfolded protein response by modulating O-GlcNAc levels of total proteins, in response to insulin resistance. Therefore, it can be a potential therapeutic target to prevent insulin resistance and endoplasmic reticulum stress.

Keywords:  3T3-L1; Adipocyte; Endoplasmic reticulum stress; Insulin resistance; O-GlcNAc; Type 2 diabetes; Unfolded protein response

DOI:  https://doi.org/10.1007/s11033-020-05665-3
Nat Chem Biol. 2020 Jul 20.

Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.

Julia M D Grandjean, Aparajita Madhavan, Lauren Cech, Bryan O Seguinot, Ryan J Paxman, Emery Smith, Louis Scampavia, Evan T Powers, Christina B Cooley, Lars Plate, Timothy P Spicer, Jeffery W Kelly, R Luke Wiseman.

Activation of the IRE1/XBP1s signaling arm of the unfolded protein response (UPR) is a promising strategy to correct defects in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. However, no pharmacologic activators of this pathway identified to date are suitable for ER proteostasis remodeling through selective activation of IRE1/XBP1s signaling. Here, we use high-throughput screening to identify non-toxic compounds that induce ER proteostasis remodeling through IRE1/XBP1s activation. We employ transcriptional profiling to stringently confirm that our prioritized compounds selectively activate IRE1/XBP1s signaling without activating other cellular stress-responsive signaling pathways. Furthermore, we demonstrate that our compounds improve ER proteostasis of destabilized variants of amyloid precursor protein (APP) through an IRE1-dependent mechanism and reduce APP-associated mitochondrial toxicity in cellular models. These results establish highly selective IRE1/XBP1s activating compounds that can be widely employed to define the functional importance of IRE1/XBP1s activity for ER proteostasis regulation in the context of health and disease.

DOI: https://doi.org/10.1038/s41589-020-0584-z
Nat Commun. 2020 Jul 20. 11(1): 3645

PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.

Yael Elbaz-Alon, Yuting Guo, Nadav Segev, Michal Harel, Daniel E Quinnell, Tamar Geiger, Ori Avinoam, Dong Li, Jodi Nunnari.

Endosomes are compositionally dynamic organelles that regulate signaling, nutrient status and organelle quality by specifying whether material entering the cells will be shuttled back to the cell surface or degraded by the lysosome. Recently, membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and endosomes have emerged as important players in endosomal protein sorting, dynamics and motility. Here, we show that PDZD8, a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain-containing ER transmembrane protein, utilizes distinct domains to interact with Rab7-GTP and the ER transmembrane protein Protrudin and together these components localize to an ER-late endosome MCS. At these ER-late endosome MCSs, mitochondria are also recruited to form a three-way contact. Thus, our data indicate that PDZD8 is a shared component of two distinct MCSs and suggest a role for SMP-mediated lipid transport in the regulation of endosome function.

DOI: https://doi.org/10.1038/s41467-020-17451-7
Nat Immunol. 2020 Aug;21(8): 868-879

STEEP mediates STING ER exit and activation of signaling.

Bao-Cun Zhang, Ramya Nandakumar, Line S Reinert, Jinrong Huang, Anders Laustsen, Zong-Liang Gao, Cheng-Long Sun, Søren Beck Jensen, Anne Troldborg, Sonia Assil, Martin F Berthelsen, Carsten Scavenius, Yan Zhang, Samuel J Windross, David Olagnier, Thaneas Prabakaran, Chiranjeevi Bodda, Ryo Narita, Yujia Cai, Cong-Gang Zhang, Harald Stenmark, Christine M Doucet, Takeshi Noda, Zheng Guo, Raphaela Goldbach-Mansky, Rune Hartmann, Zhijian J Chen, Jan J Enghild, Rasmus O Bak, Martin K Thomsen, Søren R Paludan.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

DOI: https://doi.org/10.1038/s41590-020-0730-5
J Cell Sci. 2020 Jul 21. pii: jcs.245357. [Epub ahead of print]

TMEM147 interacts with Lamin B Receptor, regulates its localization and levels, and affects cholesterol homeostasis.

Andri Christodoulou, Giannis Maimaris, Andri Makrigiorgi, Evelina Charidemou, Christian Lüchtenborg, Antonis Ververis, Renos Georgiou, Carsten W Lederer, Christof Haffner, Britta Brügger, Niovi Santama.

  The structurally and functionally complex Endoplasmic Reticulum (ER) hosts critical processes including lipid synthesis. Here, we focus on the functional characterization of transmembrane protein TMEM147 and report that it localizes at the ER and nuclear envelope in HeLa cells. Silencing of TMEM147 drastically reduces the level of lamin B receptor (LBR) at the Inner Nuclear Membrane and results in mistargeting of LBR to the ER. LBR possesses a modular structure and corresponding bifunctionality in heterochromatin organization, via its N-terminus, and cholesterol biosynthesis, via its sterol-reductase C-terminal domain. We show that TMEM147 physically interacts with LBR and the C-terminus of LBR is essential for their functional interaction. We find that TMEM147 also physically interacts with key sterol reductase DHCR7 in cholesterol biosynthesis. Similar to LBR, TMEM147 downregulation results in sharp decline of DHCR protein levels and co-ordinate transcriptional decrease of LBR and DHCR7 expression. Consistently, lipidomic analysis upon TMEM147 silencing identified changes in cellular cholesterol levels, cholesteryl ester levels and profile, and in cellular cholesterol uptake, raising the possibility that TMEM147 is an important new regulator of cholesterol homeostasis in cells.

Keywords:  Cholesterol; ER transmembrane proteins; LBR; Sterol reductases

DOI:  https://doi.org/10.1242/jcs.245357
Biomolecules. 2020 Jul 22. pii: E1090. [Epub ahead of print]10(8):

The UPR in Neurodegenerative Disease: Not Just an Inside Job.

Anna Maria van Ziel, Wiep Scheper.

  Neurons are highly specialized cells that continuously and extensively communicate with other neurons, as well as glia cells. During their long lifetime, the post-mitotic neurons encounter many stressful situations that can disrupt protein homeostasis (proteostasis). The importance of tight protein quality control is illustrated by neurodegenerative disorders where disturbed neuronal proteostasis causes neuronal dysfunction and loss. For their unique function, neurons require regulated and long-distance transport of membrane-bound cargo and organelles. This highlights the importance of protein quality control in the neuronal endomembrane system, to which the unfolded protein response (UPR) is instrumental. The UPR is a highly conserved stress response that is present in all eukaryotes. However, recent studies demonstrate the existence of cell-type-specific aspects of the UPR, as well as cell non-autonomous UPR signaling. Here we discuss these novel insights in view of the complex cellular architecture of the brain and the implications for neurodegenerative diseases.

Keywords:  cell non-autonomous; neurodegenerative disease; proteostasis; unconventional secretion; unfolded protein response

DOI:  https://doi.org/10.3390/biom10081090
Biochemistry. 2020 Jul 21.

The ATF3 Transcription Factor Is a Short-Lived Substrate of the Arg/N-Degron Pathway.

Tri T M Vu, Alexander Varshavsky.

The Arg/N-degron pathway targets proteins for degradation by recognizing their specific N-terminal residues or, alternatively, their non-N-terminal degrons. In mammals, this pathway is mediated by the UBR1, UBR2, UBR4, and UBR5 E3 ubiquitin ligases, and by the p62 regulator of autophagy. UBR1 and UBR2 are sequelogous, functionally overlapping, and dominate the targeting of Arg/N-degron substrates in examined cell lines. We constructed, here, mouse strains in which the double mutant [UBR1-/- UBR2-/-] genotype can be induced conditionally, in adult mice. We also constructed human [UBR1-/- UBR2-/-] HEK293T cell lines that unconditionally lack UBR1/UBR2. ATF3 is a basic leucine zipper transcription factor that regulates hundreds of genes and can act as either a repressor or an activator of transcription. Using the above double-mutant mice and human cells, we found that the levels of endogenous, untagged ATF3 were significantly higher in both of these [UBR1-/- UBR2-/-] settings than in wild-type cells. We also show, through chase-degradation assays with [UBR1-/- UBR2-/-] and wild-type human cells, that the Arg/N-degron pathway mediates a large fraction of ATF3 degradation. Furthermore, we used split-ubiquitin and another protein interaction assay to detect the binding of ATF3 to both UBR1 and UBR2, in agreement with the UBR1/UBR2-mediated degradation of endogenous ATF3. Full-length 24 kDa ATF3 binds to ∼100 kDa fragments of 200 kDa UBR1 and UBR2 but does not bind (in the setting of interaction assays) to full-length UBR1/UBR2. These and other binding patterns, whose mechanics remain to be understood, may signify a conditional (regulated) degradation of ATF3 by the Arg/N-degron pathway.

DOI: https://doi.org/10.1021/acs.biochem.0c00514
PLoS Biol. 2020 Jul 20. 18(7): e3000606

Proteome-wide identification of HSP70/HSC70 chaperone clients in human cells.

Seung W Ryu, Rose Stewart, D Chase Pectol, Nicolette A Ender, Oshadi Wimalarathne, Ji-Hoon Lee, Carlos P Zanini, Antony Harvey, Jon M Huibregtse, Peter Mueller, Tanya T Paull.

The 70 kDa heat shock protein (HSP70) family of chaperones are the front line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here, we demonstrate quantitative identification of HSP70 and 71 kDa heat shock cognate (HSC70) clients using a ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely nonoverlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides, but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase 1 (SOD1) mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-activated interaction trap (UBAIT) fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.

DOI: https://doi.org/10.1371/journal.pbio.3000606
Cells. 2020 Jul 16. pii: E1699. [Epub ahead of print]9(7):

Regulation of Histone Ubiquitination in Response to DNA Double Strand Breaks.

Lanni Aquila, Boyko S Atanassov.

  Eukaryotic cells are constantly exposed to both endogenous and exogenous stressors that promote the induction of DNA damage. Of this damage, double strand breaks (DSBs) are the most lethal and must be efficiently repaired in order to maintain genomic integrity. Repair of DSBs occurs primarily through one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these pathways is in part regulated by histone post-translational modifications (PTMs) including ubiquitination. Ubiquitinated histones not only influence transcription and chromatin architecture at sites neighboring DSBs but serve as critical recruitment platforms for repair machinery as well. The reversal of these modifications by deubiquitinating enzymes (DUBs) is increasingly being recognized in a number of cellular processes including DSB repair. In this context, DUBs ensure proper levels of ubiquitin, regulate recruitment of downstream effectors, dictate repair pathway choice, and facilitate appropriate termination of the repair response. This review outlines the current understanding of histone ubiquitination in response to DSBs, followed by a comprehensive overview of the DUBs that catalyze the removal of these marks.

Keywords:  DNA damage response; DSBs; DUBs; deubiquitinases; deubiquitinating enzymes; double strand break repair; histones; ubiquitination

DOI:  https://doi.org/10.3390/cells9071699
Virol J. 2020 Jul 23. 17(1): 112

The role of host eIF2α in viral infection.

Yuanzhi Liu, Mingshu Wang, Anchun Cheng, Qiao Yang, Ying Wu, Renyong Jia, Mafeng Liu, Dekang Zhu, Shun Chen, Shaqiu Zhang, Xin-Xin Zhao, Juan Huang, Sai Mao, Xumin Ou, Qun Gao, Yin Wang, Zhiwen Xu, Zhengli Chen, Ling Zhu, Qihui Luo, Yunya Liu, Yanling Yu, Ling Zhang, Bin Tian, Leichang Pan, Mujeeb Ur Rehman, Xiaoyue Chen.

   BACKGROUND: eIF2α is a regulatory node that controls protein synthesis initiation by its phosphorylation or dephosphorylation. General control nonderepressible-2 (GCN2), protein kinase R-like endoplasmic reticulum kinase (PERK), double-stranded RNA (dsRNA)-dependent protein kinase (PKR) and heme-regulated inhibitor (HRI) are four kinases that regulate eIF2α phosphorylation.
MAIN BODY: In the viral infection process, dsRNA or viral proteins produced by viral proliferation activate different eIF2α kinases, resulting in eIF2α phosphorylation, which hinders ternary tRNAMet-GTP-eIF2 complex formation and inhibits host or viral protein synthesis. The stalled messenger ribonucleoprotein (mRNP) complex aggregates under viral infection stress to form stress granules (SGs), which encapsulate viral RNA and transcription- and translation-related proteins, thereby limiting virus proliferation. However, many viruses have evolved a corresponding escape mechanism to synthesize their own proteins in the event of host protein synthesis shutdown and SG formation caused by eIF2α phosphorylation, and viruses can block the cell replication cycle through the PERK-eIF2α pathway, providing a favorable environment for their own replication. Subsequently, viruses can induce host cell autophagy or apoptosis through the eIF2α-ATF4-CHOP pathway.
CONCLUSIONS: This review summarizes the role of eIF2α in viral infection to provide a reference for studying the interactions between viruses and hosts.

Keywords:  Autophagy/apoptosis; Cell replication cycle; General translation inhibition; Stress granule; Virus; eIF2α

DOI:  https://doi.org/10.1186/s12985-020-01362-6
Autophagy. 2020 Jul 21.

PtdIns4P restriction by hydrolase SAC1 decides specific fusion of autophagosomes with lysosomes.

Hongjun Zhang, Jiao Zhou, Peng Xiao, Yong Lin, Xin Gong, Shiyan Liu, Qingjia Xu, Minjin Wang, Haiyan Ren, Mengji Lu, Yuan Wang, Jing Zhu, Zhiping Xie, Huihui Li, Kefeng Lu.

  Biogenesis of autophagosomes is the early step of macroautophagy/autophagy and requires membrane acquisition mainly from ER-Golgi-sourced precursor vesicles. Matured autophagosomes fuse with lysosomes for final degradation. However, how this selective fusion is determined remains elusive. Here, we identified Sac1 by a high throughput screen in Saccharomyces cerevisiae to show it was critical for autophagosome-lysosome fusion through its PtdIns4P phosphatase activity. Sac1 deficiency caused a dramatic increase of PtdIns4P at early Golgi apparatus and abnormal incorporation of PtdIns4P into Atg9 vesicles and autophagosomes, which caused failure to recruit SNARE proteins for autophagosome fusion with vacuoles. Sac1 function in autophagy was highly conserved from yeast to mammalian cells. Our work thus suggested that correct upstream lipid incorporation was important for downstream fusion step of autophagy and that Sac1 played a critical and ancient role in this surveillance of lipid integration.

Keywords:  Atg9; ER; Golgi; PtdIns3P; PtdIns4P; SNARE; Sac1; autophagosome-lysosome fusion; hydrolase

DOI:  https://doi.org/10.1080/15548627.2020.1796321
Neuropathology. 2020 Jul 19.

Praja1 RING-finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP-43 aggregate formation.

Kazuhiko Watabe, Yoichiro Kato, Miho Sakuma, Makiko Murata, Motoko Niida-Kawaguchi, Taro Takemura, Nobutaka Hanagata, Mari Tada, Akiyoshi Kakita, Noriyuki Shibata.

  Transactivation response DNA-binding protein of 43 kDa (TDP-43) is a major constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We have previously shown neuronal cytoplasmic aggregate formation induced by recombinant adenoviruses expressing human wild-type and C-terminal fragment (CTF) TDP-43 under the condition of proteasome inhibition in vitro and in vivo. In the present study, we demonstrated that the formation of the adenoviral TDP-43 aggregates was markedly suppressed in rat neural stem cell-derived neuronal cells by co-infection of an adenovirus expressing heat shock transcription factor 1 (HSF1), a master regulator of heat shock response. We performed DNA microarray analysis and searched several candidate molecules, located downstream of HSF1, which counteract TDP-43 aggregate formation. Among these, we identified Praja 1 RING-finger E3 ubiquitin ligase (PJA1) as a suppressor of phosphorylation and aggregate formation of TDP-43. Co-immunoprecipitation assay revealed that PJA1 binds to CTF TDP-43 and the E2-conjugating enzyme UBE2E3. PJA1 also suppressed formation of cytoplasmic phosphorylated TDP-43 aggregates in mouse facial motor neurons in vivo. Furthermore, phosphorylated TDP-43 aggregates were detected in PJA1-immunoreactive human ALS motor neurons. These results indicate that PJA1 is one of the principal E3 ubiquitin ligases for TDP-43 to counteract its aggregation propensity and could be a potential therapeutic target for ALS and FTLD.

Keywords:  Praja1; TDP-43; adenovirus; amyotrophic lateral sclerosis; frontotemporal lobar degeneration

DOI:  https://doi.org/10.1111/neup.12694
Nat Microbiol. 2020 Jul 20.

Inhibition of the integrated stress response by viral proteins that block p-eIF2-eIF2B association.

Huib H Rabouw, Linda J Visser, Tim C Passchier, Martijn A Langereis, Fan Liu, Piero Giansanti, Arno L W van Vliet, José G Dekker, Susanne G van der Grein, Jesús G Saucedo, Aditya A Anand, Mikael E Trellet, Alexandre M J J Bonvin, Peter Walter, Albert J R Heck, Raoul J de Groot, Frank J M van Kuppeveld.

Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.

DOI: https://doi.org/10.1038/s41564-020-0759-0
Nucleic Acids Res. 2020 Jul 25. pii: gkaa608. [Epub ahead of print]

Gcn2 eIF2α kinase mediates combinatorial translational regulation through nucleotide motifs and uORFs in target mRNAs.

Yuji Chikashige, Hiroaki Kato, Mackenzie Thornton, Whitney Pepper, Madelyn Hilgers, Ariana Cecil, Izumi Asano, Haana Yamada, Chie Mori, Cheyenne Brunkow, Carter Moravek, Takeshi Urano, Chingakham Ranjit Singh, Katsura Asano.

The protein kinase Gcn2 is a central transducer of nutritional stress signaling important for stress adaptation by normal cells and the survival of cancer cells. In response to nutrient deprivation, Gcn2 phosphorylates eIF2α, thereby repressing general translation while enhancing translation of specific mRNAs with upstream ORFs (uORFs) situated in their 5'-leader regions. Here we performed genome-wide measurements of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysome analyses were combined with microarray measurements to identify gene transcripts whose translation was up-regulated in response to the stress in a Gcn2-dependent manner. We determined that translation is reprogrammed to enhance RNA metabolism and chromatin regulation and repress ribosome synthesis. Interestingly, translation of intron-containing mRNAs was up-regulated. The products of the regulated genes include additional eIF2α kinase Hri2 amplifying the stress signaling and Gcn5 histone acetyl transferase and transcription factors, together altering genome-wide transcription. Unique dipeptide-coding uORFs and nucleotide motifs, such as '5'-UGA(C/G)GG-3', are found in 5' leader regions of regulated genes and shown to be responsible for translational control.

DOI: https://doi.org/10.1093/nar/gkaa608
J Virol. 2020 Jul 22. pii: JVI.00874-20. [Epub ahead of print]

N-linked glycan sites on the influenza NA head domain are required for efficient IAV incorporation and replication.

Henrik Östbye, Jin Gao, Mira Rakic Martinez, Hao Wang, Jan-Willem de Gier, Robert Daniels.

N-linked glycans commonly contribute to secretory protein folding, sorting and signaling. For enveloped viruses such as the influenza A virus (IAV), the addition of large N-linked glycans can also prevent access to epitopes on the surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N). Sequence analysis showed that in the NA head domain of H1N1 IAVs three N-linked glycosylation sites are conserved and that a fourth site is conserved in H3N2 IAVs. Variable sites are almost exclusive to H1N1 IAVs of human origin, where the number of head glycosylation sites first increased over time and then decreased with and after the introduction of the 2009 pandemic H1N1 IAV of Eurasian swine origin. In contrast, variable sites exist in H3N2 IAVs of human and swine origin, where the number of head glycosylation sites has mainly increased over time. Analysis of IAVs carrying N1 and N2 mutants demonstrated that the N-linked glycosylation sites on the NA head domain are required for efficient virion incorporation and replication in cells and eggs. It also revealed that N1 stability is more affected by the head domain glycans, suggesting N2 is more amenable to glycan additions. Together, these results indicate that in addition to antigenicity, N-linked glycosylation sites can alter NA enzymatic stability and the NA amount in virions.IMPORTANCE N-linked glycans are transferred to secretory proteins upon entry into the endoplasmic reticulum lumen. In addition to promoting secretory protein maturation, enveloped viruses also utilize these large oligosaccharide structures to prevent access to surface antigen epitopes. Sequence analyses of the influenza A virus (IAV) surface antigen neuraminidase (NA or N) showed that the conservation of N-linked glycosylation sites on the NA enzymatic head domain differs by IAV subtype (H1N1 vs H3N2) and species of origin, with human derived IAVs possessing the most variability. Experimental analyses verified that the N-linked glycosylation sites on the NA head domain contribute to virion incorporation and replication. It also revealed that the head domain glycans affect N1 stability more than N2, suggesting N2 is more accommodating to glycan additions. These results demonstrate that in addition to antigenicity, changes in N-linked glycosylation sites can alter other properties of viral surface antigens and virions.

DOI: https://doi.org/10.1128/JVI.00874-20
Mol Cell Endocrinol. 2020 Jul 18. pii: S0303-7207(20)30248-3. [Epub ahead of print] 110948

VIP conditions human endometrial receptivity by privileging endoplasmic reticulum stress through ATF6α pathway.

E Soczewski, S Gori, D Paparini, E Grasso, L Fernández, L Gallino, Ana Schafir, M Irigoyen, T F Lobo, G Salamone, R Mattar, S Daher, C Pérez Leirós, R Ramhorst.

  Endometrial stromal cells undergo endoplasmic reticulum (ER) stress and unfolded protein response (UPR) during the decidualization linked with the inflammation and angiogenesis processes. Considering VIP (vasoactive intestinal peptide) induces the decidualization program, we studied whether modulates the ER/UPR pathways to condition both processes for embryo implantation. When Human endometrial stromal cell line (HESC) were decidualized by VIP we observed an increase expression of ATF6α, an ER stress-sensor, and UPR markers, associated with an increase in IL-1β production. Moreover, AEBSF (ATF6α -inhibitor pathway) prevented this effect and decreased the expansion index in the in vitro model of implantation. VIP-decidualized cells also favor angiogenesis accompanied by a strong downregulation in thrombospondin-1. Finally, ATF6α, VIP and VPAC2-receptor expression were reduced in endometrial biopsies from women with recurrent implantation failures in comparison with fertile. In conclusion, VIP privileged ATF6α-pathway associated with a sterile inflammatory response and angiogenesis that might condition endometrial receptivity.

Keywords:  Decidualization; Reticular stress; Sterile inflammation; Unfolded protein response; Vasoactive intestinal peptide

DOI:  https://doi.org/10.1016/j.mce.2020.110948
Genome Res. 2020 Jul 23.

Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing.

Alaaddin Bulak Arpat, Angélica Liechti, Mara De Matos, René Dreos, Peggy Janich, David Gatfield.

Translation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of nonuniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs, and nascent polypeptide structure is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.

DOI: https://doi.org/10.1101/gr.257741.119
Eur J Immunol. 2020 Jul 20.

Evidence for an involvement of the ubiquitin-like modifier ISG15 in MHC class I antigen presentation.

Tobias Held, Michael Basler, Klaus-Peter Knobeloch, Marcus Groettrup.

  The interferon stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein, that is induced by type I interferons and is conjugated to the bulk of newly synthesized polypeptides at the ribosome. ISG15 functions as an antiviral molecule possibly by being covalently conjugated to viral proteins and disturbing virus particle assembly. Here, we have investigated the effect of ISGylation on degradation and antigen presentation of viral and cellular proteins. ISGylation did not induce proteasomal degradation of bulk ISG15 target proteins neither after overexpressing ISG15 nor after induction by interferon-β (IFN-β). The MHC class I cell surface expression of splenocytes derived from ISG15-deficient mice or mice lacking the catalytic activity of the major de-ISGylating enzyme USP18 was unaltered as compared to wild type mice. Fusion of ubiquitin or FAT10 to the long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus accelerated the proteasomal degradation of NP while fusion to ISG15 did not detectably speed up NP degradation. Nevertheless, MHC-I restricted presentation of two epitopes of NP were markedly enhanced when it was fused to ISG15 similarly to fusion with ubiquitin or FAT10. Thus, we provide evidence that ISG15 can enhance the presentation of antigens on MHC-I most likely by promoting co-translational antigen processing. This article is protected by copyright. All rights reserved.

Keywords:  ISG15; MHC class I; antigen presentation; proteasome; ubiquitin-like modifier

DOI:  https://doi.org/10.1002/eji.202048646
Biomolecules. 2020 Jul 21. pii: E1082. [Epub ahead of print]10(7):

The COP9 Signalosome: A Multi-DUB Complex.

Wolfgang Dubiel, Supattra Chaithongyot, Dawadschargal Dubiel, Michael Naumann.

  The COP9 signalosome (CSN) is a signaling platform controlling the cellular ubiquitylation status. It determines the activity and remodeling of ~700 cullin-RING ubiquitin ligases (CRLs), which control more than 20% of all ubiquitylation events in cells and thereby influence virtually any cellular pathway. In addition, it is associated with deubiquitylating enzymes (DUBs) protecting CRLs from autoubiquitylation and rescuing ubiquitylated proteins from degradation. The coordination of ubiquitylation and deubiquitylation by the CSN is presumably important for fine-tuning the precise formation of defined ubiquitin chains. Considering its intrinsic DUB activity specific for deneddylation of CRLs and belonging to the JAMM family as well as its associated DUBs, the CSN represents a multi-DUB complex. Two CSN-associated DUBs, the ubiquitin-specific protease 15 (USP15) and USP48 are regulators in the NF-κB signaling pathway. USP15 protects CRL1β-TrCP responsible for IκBα ubiquitylation, whereas USP48 stabilizes the nuclear pool of the NF-κB transcription factor RelA upon TNF stimulation by counteracting CRL2SOCS1. Moreover, the CSN controls the neddylation status of cells by its intrinsic DUB activity and by destabilizing the associated deneddylation enzyme 1 (DEN1). Thus, the CSN is a master regulator at the intersection between ubiquitylation and neddylation.

Keywords:  COP9 signalosome; DEN1; DUBs; USP15; USP48; cullin-RING ubiquitin ligases; ubiquitylation

DOI:  https://doi.org/10.3390/biom10071082
Int J Oncol. 2020 Sep;57(3): 835-844

Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells.

Sangkab Sudsaward, Sasiprapa Khunchai, Chutamas Thepmalee, Aisha Othman, Thawornchai Limjindaporn, Pa-Thai Yenchitsomanus, Luciano Mutti, Marija Krstic-Demonacos, Constantinos Demonacos.

Acute lymphoblastic leukaemia (ALL) is the most frequent childhood cancer and, although it is highly treatable, resistance to therapy, toxicity and side effects remain challenging. The synthetic glucocorticoid (GC) dexamethasone (Dex) is commonly used to treat ALL, the main drawback of which is the development of resistance to this treatment. The aim of the present study was to investigate potential molecular circuits mediating resistance and sensitivity to GC‑induced apoptosis in ALL. The leukaemia cell lines CEM‑C7‑14, CEM‑C1‑15 and MOLT4 treated with chloroquine (CLQ), thapsigargin (TG) and rotenone (ROT) were used to explore the roles of autophagy, endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and reactive oxygen species (ROS) generation in the response to GC treatment. ROS levels were associated with increased cell death and mitochondrial membrane potential in rotenone‑treated CEM cells. Autophagy inhibition by CLQ exhibited the strongest cytotoxic effect in GC‑resistant leukaemia. Autophagy may act as a pro‑survival mechanism in GC‑resistant leukaemia since increasing trends in beclin‑1 and microtubule‑associated protein 1 light chain 3α levels were detected in CEM‑C1‑15 and MOLT4 cells treated with Dex, whereas decreasing trends in these autophagy markers were observed in CEM‑C7‑14 cells. The intracellular protein levels of the ER stress markers glucose‑regulated protein (GRP)78 and GRP94 were stimulated by Dex only in the GC‑sensitive cells, suggesting a role of these chaperones in the GC‑mediated ALL cell death. Increased cell surface levels of GRP94 were recorded in CEM‑C7‑14 cells treated with combination of Dex with TG compared with those in cells treated with TG alone, whereas decreasing trends were observed in CEM‑C1‑15 cells under these conditions. Taken together, the results of the present study demonstrated that autophagy may be a pro‑survival mechanism in GC‑resistant leukaemia, and by modulating intracellular and surface GRP94 protein levels, Dex is involved in the regulation of ER stress/UPR‑dependent cell death and immune surveillance. These observations may be of clinical importance if confirmed in patients.

DOI: https://doi.org/10.3892/ijo.2020.5089
Fish Shellfish Immunol. 2020 Jul 19. pii: S1050-4648(20)30499-X. [Epub ahead of print]

Grouper ubiquitin-specific protease 14 promotes iridovirus replication through negatively regulating interferon response.

Liwei Huang, Ya Zhang, Jiaying Zheng, Na Ni, Qiwei Qin, Xiaohong Huang, Youhua Huang.

  Ubiquitin-specific protease 14 (USP14), one of the USP family members which belong to deubiquitinating enzymes (DUBs), plays a key role in maintaining cellular protein homeostasis by trimming ubiquitin chains from their substrates. However, the roles of USP14 in response to virus infection still remains largely unknown. In the current study, a USP14 homolog from orange spotted grouper (EcUSP14) was cloned and its roles in innate immune response were investigated. EcUSP14 was composed of 1479 base pairs encoding a 492-amino acid (aa) polypeptide. Sequence analysis indicated that EcUSP14 shared 96.14% and 81.30% identity to USP14 of bicolor damselfish (Stegastes partitus) and humans (homo sapiens), respectively. EcUSP14 contains conserved ubiquitin-like (UBL) domain (aa 3-76) and peptidase-C19A domain (aa 106-481). In response to Singapore grouper iridovirus (SGIV) infection in vitro, EcUSP14 was significantly up-regulated. Subcellular localization showed that EcUSP14 was predominantly localized in the cytoplasm of grouper spleen (GS) cells and mostly co-localized with the viral assembly sites after SGIV infection. The ectopic expression of EcUSP14 significantly promoted the replication of SGIV, as demonstrated by the accelerated progression of severity of cytopathic eﬀect (CPE), the increased viral gene transcription and viral protein synthesis during infection. Consistently, treatment with IU1, a USP14 specific inhibitor, significantly inhibited the replication of SGIV, suggesting that USP14 function as a pro-viral factor during SGIV replication. Further analysis showed that EcUSP14 overexpression decreased the promoter activities of interferon (IFN)-1, IFN-3, IFN-stimulated response element (ISRE), and nuclear factor of kappa B (NF-κB). Furthermore, the ectopic expression of EcUSP14 decreased the activities of IFN-1 promoter evoked by TANK-binding kinase (TBK)-1 and melanoma diﬀerentiation-associated protein (MDA)-5, but not stimulator of interferon genes (STING). Thus, we speculated that EcUSP14 facilitated virus replication by negatively regulating the IFN response. Taken together, our results firstly demonstrated that fish USP14 functioned as a pro-viral factor by negatively regulating interferon response against virus infection.

Keywords:  Grouper; Interferon; SGIV; USP14; Virus replication

DOI:  https://doi.org/10.1016/j.fsi.2020.07.015
Nat Metab. 2020 Apr;2(4): 318-334

NRF2 activation promotes the recurrence of dormant tumour cells through regulation of redox and nucleotide metabolism.

Douglas B Fox, Nina Marie G Garcia, Brock J McKinney, Ryan Lupo, Laura C Noteware, Rachel Newcomb, Juan Liu, Jason W Locasale, Matthew D Hirschey, James V Alvarez.

  The survival and recurrence of dormant tumour cells following therapy is a leading cause of death in cancer patients. The metabolic properties of these cells are likely distinct from those of rapidly growing tumours. Here we show that Her2 down-regulation in breast cancer cells promotes changes in cellular metabolism, culminating in oxidative stress and compensatory upregulation of the antioxidant transcription factor, NRF2. NRF2 is activated during dormancy and in recurrent tumours in animal models and breast cancer patients with poor prognosis. Constitutive activation of NRF2 accelerates recurrence, while suppression of NRF2 impairs it. In recurrent tumours, NRF2 signalling induces a transcriptional metabolic reprogramming to re-establish redox homeostasis and upregulate de novo nucleotide synthesis. The NRF2-driven metabolic state renders recurrent tumour cells sensitive to glutaminase inhibition, which prevents reactivation of dormant tumour cells in vitro, suggesting that NRF2-high dormant and recurrent tumours may be targeted. These data provide evidence that NRF2-driven metabolic reprogramming promotes the recurrence of dormant breast cancer.

Keywords:  Breast cancer recurrence; Her2; NRF2; ROS; Residual disease; Tumor metabolism

DOI:  https://doi.org/10.1038/s42255-020-0191-z
Haematologica. 2020 Jul 23. pii: haematol.2019.237321. [Epub ahead of print]

Endoplasmic reticulum stress controls iron metabolism through TMPRSS6 repression and hepcidin mRNA stabilization by RNA-binding protein HuR.

Audrey Belot, Ophélie Gourbeyre, Anais Palin, Aude Rubio, Amélie Largounez, Céline Besson-Fournier, Chloé Latour, Megane Lorgouilloux, Inka Gallitz, Alexandra Montagner, Arnaud Polizzi, Marion Régnier, Sarra Smati, An-Sheng Zhang, Manuel D Diaz-Munoz, Andrea U Steinbicker, Hervé Guillou, Marie-Paule Roth, Hélène Coppin, Delphine Meynard.



Keywords:  ER stress; Hepcidin; Iron Metabolism; Matriptase-2

DOI:  https://doi.org/10.3324/haematol.2019.237321
Oxid Med Cell Longev. 2020 ;2020 2067959

Mitogen- and Stress-Activated Protein Kinase 1 Mediates Alcohol-Upregulated Transcription of Brf1 and tRNA Genes to Cause Phenotypic Alteration.

Mingen Lin, Chenghao Huang, Wenfeng Ren, Jun Chen, Ningshao Xia, Shuping Zhong.

Upregulation of Brf1 (TFIIB-related factor 1) and Pol III gene (RNA polymerase III-dependent gene, such as tRNAs and 5S rRNA) activities is associated with cell transformation and tumor development. Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, and cirrhosis, which enhances the risk of HCC development. However, the mechanism of alcohol-promoted HCC remains to be explored. We have designed the complementary research system, which is composed of cell lines, an animal model, human samples, and experiments in vivo and in vitro, to carry out this project by using molecular biological, biochemical, and cellular biological approaches. It is a unique system to explore the mechanism of alcohol-associated HCC. Our results indicate that alcohol upregulates Brf1 and Pol III gene (tRNAs and 5S rRNA) transcription in primary mouse hepatocytes, immortalized mouse hepatocyte-AML-12 cells, and engineered human HepG2-ADH cells. Alcohol activates MSK1 to upregulate expression of Brf1 and Pol III genes, while inhibiting MSK1 reduces transcription of Brf1 and Pol III genes in alcohol-treated cells. The inhibitor of MSK1, SB-747651A, decreases the rates of cell proliferation and colony formation. Alcohol feeding promotes liver tumor development of the mouse. These results, for the first time, show the identification of the alcohol-response promoter fragment of the Pol III gene key transcription factor, Brf1. Our studies demonstrate that Brf1 expression is elevated in HCC tumor tissues of mice and humans. Alcohol increases cellular levels of Brf1, resulting in enhancement of Pol III gene transcription in hepatocytes through MSK1. Our mechanism analysis has demonstrated that alcohol-caused high-response fragment of the Brf1 promoter is at p-382/+109bp. The MSK1 inhibitor SB-747651A is an effective reagent to repress alcohol-induced cell proliferation and colony formation, which is a potential pharmaceutical agent. Developing this inhibitor as a therapeutic approach will benefit alcohol-associated HCC patients.

DOI: https://doi.org/10.1155/2020/2067959
Cell Rep. 2020 Jul 21. pii: S2211-1247(20)30907-4. [Epub ahead of print]32(3): 107926

Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane-Proximal Regions of VAMP2 and SNAP25.

Jörg Malsam, Simon Bärfuss, Thorsten Trimbuch, Fereshteh Zarebidaki, Andreas F-P Sonnen, Klemens Wild, Andrea Scheutzow, Lukas Rohland, Matthias P Mayer, Irmgard Sinning, John A G Briggs, Christian Rosenmund, Thomas H Söllner.

  The neuronal protein complexin contains multiple domains that exert clamping and facilitatory functions to tune spontaneous and action potential-triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that, prior to fusion, the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitters in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

Keywords:  SNARE; complexin; cross-linking; membrane fusion; neurotransmission; synaptotagmin; syntaxin

DOI:  https://doi.org/10.1016/j.celrep.2020.107926
Autophagy. 2020 Jul 18. 1-3

A nuclear role for Atg8-family proteins.

Anne-Claire Jacomin, Stavroula Petridi, Marisa Di Monaco, Ioannis P Nezis.

  Despite the growing evidence that the macroautophagy/autophagy-related protein LC3 is localized in the nucleus, why and how it is targeted to the nucleus are poorly understood. In our recent study, we found that transcription factor seq (sequoia) interacts via its LIR motif with Atg8a, the Drosophila homolog of LC3, to negatively regulate the transcription of autophagy genes. Atg8a was found to also interact with the nuclear acetyltransferase complex subunit YL-1 and deacetylase Sirt2. Modulation of the acetylation status of Atg8a by YL-1 and Sirt2 affects the interaction between seq and Atg8a, and controls the induction of autophagy. Our work revealed a novel nuclear role for Atg8a, which is linked with the transcriptional regulation of autophagy genes.

Keywords:  Acetylation; LC3/Atg8; LIR motif; autophagy; nucleus; transcription

DOI:  https://doi.org/10.1080/15548627.2020.1794356
Psychoneuroendocrinology. 2020 Jul 11. pii: S0306-4530(20)30222-5. [Epub ahead of print]120 104800

SUMO E3 ligase PIAS1 is a potential biomarker indicating stress susceptibility.

Hsiao-Yun Lin, Yu-Shu Liu, Ching-Ya Huang, Flurin Cathomas, Kalena Liu, Jun Wang, Han-Tsung Cheng, Sheng-Wei Lai, Yu-Ching Liu, Chao-Jung Chen, Chingju Lin, Dah-Yuu Lu.

  Prior studies suggest that individual differences in stress responses contribute to the pathogenesis of neuropsychiatric disorders. In the present study, we investigated the role of small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) in mediating stress responses to chronic social defeat stress (CSDS). We found that mRNA and protein levels of PIAS 1 were decreased in the hippocampus of high-susceptibility (HS) mice but not in low-susceptibility (LS) mice after CSDS. Local overexpression of PIAS1 in the hippocampus followed by CSDS exposure promoted stress resilience by attenuating social avoidance and improving anxiety-like behaviors. Viral-mediated gene transfer to generate a conditional knockdown of PIAS1 in the hippocampus promoted social avoidance and stress vulnerability after subthreshold microdefeat. HS mice displayed decreased levels of glucocorticoid receptor (GR) expression, and GR SUMOylation in the hippocampus was associated with stress vulnerability. Furthermore, cytokine/chemokine levels were changed predominantly in the hippocampus of HS mice. These results suggest that hippocampal PIAS1 plays a role in the regulation of stress susceptibility by post-translational modification of GRs.

Keywords:  Chronic social defeat stress (CSDS); Glucocorticoid receptors (GR); PIAS1; SUMOylation; Susceptibility

DOI:  https://doi.org/10.1016/j.psyneuen.2020.104800
Cell Stress Chaperones. 2020 Jul 21.

BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes.

Laura Mediani, Veronica Galli, Arianna Dorotea Carrà, Ilaria Bigi, Jonathan Vinet, Massimo Ganassi, Francesco Antoniani, Tatiana Tiago, Marco Cimino, Daniel Mateju, Cristina Cereda, Orietta Pansarasa, Simon Alberti, Jessica Mandrioli, Serena Carra.

  Stress granules (SGs) are dynamic ribonucleoprotein granules induced by environmental stresses. They play an important role in the stress response by integrating mRNA stability, translation, and signaling pathways. Recent work has connected SG dysfunction to neurodegenerative diseases. In these diseases, SG dynamics are impaired because of mutations in SG proteins or protein quality control factors. Impaired SG dynamics and delayed SG dissolution have also been observed for SGs that accumulate misfolding-prone defective ribosomal products (DRiPs). DRiP accumulation inside SGs is controlled by a surveillance system referred to as granulostasis and encompasses the molecular chaperones VCP and the HSPB8-BAG3-HSP70 complex. BAG3 is a member of the BAG family of proteins, which includes five additional members. One of these proteins, BAG6, is functionally related to BAG3 and able to assist degradation of DRiPs. However, whether BAG6 is involved in granulostasis is unknown. We report that BAG6 is not recruited into SGs induced by different types of stress, nor does it affect SG dynamics. BAG6 also does not replace BAG3's function in SG granulostasis. We show that BAG3 and BAG6 target different subsets of DRiPs, and BAG3 binding to DRiPs is mediated by HSPB8 and HSP70. Our data support the idea that SGs are sensitive to BAG3-HSP70-bound DRiPs but not to BAG6-bound DRiPs. Additionally, only BAG3 is strongly upregulated in the stress recovery phase, when SGs dissolve. These data exclude a role for BAG6 in granulostasis and point to a more specialized function in the clearance of a specific subset of DRiPs.

Keywords:  BAG proteins; Co-chaperones; Newly synthesized proteins; Protein clearance; Stress granule dynamics

DOI:  https://doi.org/10.1007/s12192-020-01141-w