bims-tunefa Biomed News
on Tumor necrosis factor superfamily and post-translational modifications
Issue of 2020‒06‒21
thirty papers selected by
John Silke
Walter and Eliza Hall Institute of Medical Research


  1. Nat Commun. 2020 Jun 19. 11(1): 3151
    Samson AL, Zhang Y, Geoghegan ND, Gavin XJ, Davies KA, Mlodzianoski MJ, Whitehead LW, Frank D, Garnish SE, Fitzgibbon C, Hempel A, Young SN, Jacobsen AV, Cawthorne W, Petrie EJ, Faux MC, Shield-Artin K, Lalaoui N, Hildebrand JM, Silke J, Rogers KL, Lessene G, Hawkins ED, Murphy JM.
      Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies.
    DOI:  https://doi.org/10.1038/s41467-020-16887-1
  2. Biomolecules. 2020 Jun 14. pii: E903. [Epub ahead of print]10(6):
    Leboeuf D, Pyatkov M, Zatsepin TS, Piatkov K.
      Recognition of danger signals by a cell initiates a powerful cascade of events generally leading to inflammation. Inflammatory caspases and several other proteases become activated and subsequently cleave their target proinflammatory mediators. The irreversible nature of this process implies that the newly generated proinflammatory fragments need to be sequestered, inhibited, or degraded in order to cancel the proinflammatory program or prevent chronic inflammation. The Arg/N-degron pathway is a ubiquitin-dependent proteolytic pathway that specifically degrades protein fragments bearing N-degrons, or destabilizing residues, which are recognized by the E3 ligases of the pathway. Here, we report that the Arg/N-degron pathway selectively degrades a number of proinflammatory fragments, including some activated inflammatory caspases, contributing in tuning inflammatory processes. Partial ablation of the Arg/N-degron pathway greatly increases IL-1β secretion, indicating the importance of this ubiquitous pathway in the initiation and resolution of inflammation. Thus, we propose a model wherein the Arg/N-degron pathway participates in the control of inflammation in two ways: in the generation of inflammatory signals by the degradation of inhibitory anti-inflammatory domains and as an "off switch" for inflammatory responses through the selective degradation of proinflammatory fragments.
    Keywords:  Arg/N-degron pathway; UBR-ubiquitin ligases; inflammation; inflammatory caspases; proteolysis; ubiquitin
    DOI:  https://doi.org/10.3390/biom10060903
  3. J Cell Biol. 2020 Jul 06. pii: e201908087. [Epub ahead of print]219(7):
    Tang D, Sandoval W, Lam C, Haley B, Liu P, Xue D, Roy D, Patapoff T, Louie S, Snedecor B, Misaghi S.
      Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.
    DOI:  https://doi.org/10.1083/jcb.201908087
  4. J Cell Biol. 2020 Jul 06. pii: e202004003. [Epub ahead of print]219(7):
    Weavers H, Martin P.
      Tissue damage triggers a rapid and robust inflammatory response in order to clear and repair a wound. Remarkably, many of the cell biology features that underlie the ability of leukocytes to home in to sites of injury and to fight infection-most of which are topics of intensive current research-were originally observed in various weird and wonderful translucent organisms over a century ago by Elie Metchnikoff, the "father of innate immunity," who is credited with discovering phagocytes in 1882. In this review, we use Metchnikoff's seminal lectures as a starting point to discuss the tremendous variety of cell biology features that underpin the function of these multitasking immune cells. Some of these are shared by other cell types (including aspects of motility, membrane trafficking, cell division, and death), but others are more unique features of innate immune cells, enabling them to fulfill their specialized functions, such as encapsulation of invading pathogens, cell-cell fusion in response to foreign bodies, and their self-sacrifice as occurs during NETosis.
    DOI:  https://doi.org/10.1083/jcb.202004003
  5. Immunity. 2020 Jun 16. pii: S1074-7613(20)30213-2. [Epub ahead of print]52(6): 942-956
    Nutt SL, Chopin M.
      Dendritic cells (DCs) are the sentinels of the immune system, sensing a diverse array of pathogens to stimulate a robust and appropriate immune response. To initiate responses to highly disparate challenges, DCs have diversified into multiple phenotypically, anatomically, and functionally distinct cell types. As a result of the application of new single-cell technologies, the full extent of this diversity, as well as the developmental relationships of the DC lineages, is currently undergoing reassessment. Here, we review the cellular and molecular evidence that underpins current models of DC differentiation and functional diversification in the murine and human systems. We discuss these models in the context of the diversity revealed by single-cell studies and propose that understanding DC identity will require defining the regulatory interactions that control gene expression in these cells.
    DOI:  https://doi.org/10.1016/j.immuni.2020.05.005
  6. Nat Commun. 2020 Jun 19. 11(1): 3060
    Davies KA, Fitzgibbon C, Young SN, Garnish SE, Yeung W, Coursier D, Birkinshaw RW, Sandow JJ, Lehmann WIL, Liang LY, Lucet IS, Chalmers JD, Patrick WM, Kannan N, Petrie EJ, Czabotar PE, Murphy JM.
      The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL's conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells. Here, we examine the divergence of activation mechanisms among nine vertebrate MLKL orthologues, revealing remarkable specificity of mouse and human RIPK3 for MLKL orthologues. Pig MLKL can restore necroptotic signaling in human cells; while horse and pig, but not rat, MLKL can reconstitute the mouse pathway. This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. These studies identify important differences in necroptotic signaling between species, and suggest that, more broadly, divergent regulatory mechanisms may exist among orthologous pseudoenzymes.
    DOI:  https://doi.org/10.1038/s41467-020-16823-3
  7. Proc Natl Acad Sci U S A. 2020 Jun 17. pii: 201911154. [Epub ahead of print]
    Zajc CU, Dobersberger M, Schaffner I, Mlynek G, Pühringer D, Salzer B, Djinović-Carugo K, Steinberger P, De Sousa Linhares A, Yang NJ, Obinger C, Holter W, Traxlmayr MW, Lehner M.
      Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.
    Keywords:  CAR T cell; CID; alternative scaffold; lipocalin; protein engineering
    DOI:  https://doi.org/10.1073/pnas.1911154117
  8. Immunity. 2020 Jun 16. pii: S1074-7613(20)30216-8. [Epub ahead of print]52(6): 890-892
    Amaral MP, Bortoluci KR.
      Caspase-8 is a master regulator of cell death pathways, although its regulation during inflammation remains elusive. Using elegant mouse genetic approaches, Schwarzer et al. and Tummers et al. revealed the importance of FADD in regulating caspase-8-mediated inflammatory responses and gut homeostasis.
    DOI:  https://doi.org/10.1016/j.immuni.2020.05.008
  9. Nat Struct Mol Biol. 2020 Jun 15.
    Wu T, Yoon H, Xiong Y, Dixon-Clarke SE, Nowak RP, Fischer ES.
      Controlled perturbation of protein activity is essential to study protein function in cells and living organisms. Small molecules that hijack the cellular protein ubiquitination machinery to selectively degrade proteins of interest, so-called degraders, have recently emerged as alternatives to selective chemical inhibitors, both as therapeutic modalities and as powerful research tools. These systems offer unprecedented temporal and spatial control over protein function. Here, we review recent developments in this field, with a particular focus on the use of degraders as research tools to interrogate complex biological problems.
    DOI:  https://doi.org/10.1038/s41594-020-0438-0
  10. Nat Commun. 2020 Jun 19. 11(1): 3150
    Hildebrand JM, Kauppi M, Majewski IJ, Liu Z, Cox AJ, Miyake S, Petrie EJ, Silk MA, Li Z, Tanzer MC, Brumatti G, Young SN, Hall C, Garnish SE, Corbin J, Stutz MD, Di Rago L, Gangatirkar P, Josefsson EC, Rigbye K, Anderton H, Rickard JA, Tripaydonis A, Sheridan J, Scerri TS, Jackson VE, Czabotar PE, Zhang JG, Varghese L, Allison CC, Pellegrini M, Tannahill GM, Hatchell EC, Willson TA, Stockwell D, de Graaf CA, Collinge J, Hilton A, Silke N, Spall SK, Chau D, Athanasopoulos V, Metcalf D, Laxer RM, Bassuk AG, Darbro BW, Fiatarone Singh MA, Vlahovich N, Hughes D, Kozlovskaia M, Ascher DB, Warnatz K, Venhoff N, Thiel J, Biben C, Blum S, Reveille J, Hildebrand MS, Vinuesa CG, McCombe P, Brown MA, Kile BT, McLean C, Bahlo M, Masters SL, Nakano H, Ferguson PJ, Murphy JM, Alexander WS, Silke J.
      MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).
    DOI:  https://doi.org/10.1038/s41467-020-16819-z
  11. JCI Insight. 2020 Jun 18. pii: 133668. [Epub ahead of print]5(12):
    Schafer MJ, Zhang X, Kumar A, Atkinson EJ, Zhu Y, Jachim S, Mazula DL, Brown AK, Berning M, Aversa Z, Kotajarvi B, Bruce CJ, Greason KL, Suri RM, Tracy RP, Cummings SR, White TA, LeBrasseur NK.
      Produced by senescent cells, the senescence-associated secretory phenotype (SASP) is a potential driver of age-related dysfunction. We tested whether circulating concentrations of SASP proteins reflect age and medical risk in humans. We first screened senescent endothelial cells, fibroblasts, preadipocytes, epithelial cells, and myoblasts to identify candidates for human profiling. We then tested associations between circulating SASP proteins and clinical data from individuals throughout the life span and older adults undergoing surgery for prevalent but distinct age-related diseases. A community-based sample of people aged 20-90 years (retrospective cross-sectional) was studied to test associations between circulating SASP factors and chronological age. A subset of this cohort aged 60-90 years and separate cohorts of older adults undergoing surgery for severe aortic stenosis (prospective longitudinal) or ovarian cancer (prospective case-control) were studied to assess relationships between circulating concentrations of SASP proteins and biological age (determined by the accumulation of age-related health deficits) and/or postsurgical outcomes. We showed that SASP proteins were positively associated with age, frailty, and adverse postsurgery outcomes. A panel of 7 SASP factors composed of growth differentiation factor 15 (GDF15), TNF receptor superfamily member 6 (FAS), osteopontin (OPN), TNF receptor 1 (TNFR1), ACTIVIN A, chemokine (C-C motif) ligand 3 (CCL3), and IL-15 predicted adverse events markedly better than a single SASP protein or age. Our findings suggest that the circulating SASP may serve as a clinically useful candidate biomarker of age-related health and a powerful tool for interventional human studies.
    Keywords:  Aging; Cellular senescence
    DOI:  https://doi.org/10.1172/jci.insight.133668
  12. ChemMedChem. 2020 Jun 17.
    Manna D, Verma S.
      PROTACs are emerging as a powerful strategy for exerting exogenous control over protein levels, allowing small molecules to exploit the ubiquitin-proteasome pathway for targeted protein degradation. This highlight focuses on the fusion of photochemistry with these bifunctional compounds which has provided a novel pathway for spatiotemporally tuning the activation of PROTACs in the form of their photocaged and photoswitchable versions. Photocaged PROTACs consist of a hindered optolabile group which detaches only on irradiation of specific wavelength, releasing the active PROTAC. These modified PROTACs are inactive in the dark. Photoswitchable PROTACs are photoisomerizable molecules with azobenzene linkages which are active in either of the cis or trans isomers and obligatorily inactive in the other. The isomers are interconvertible by irradiation with appropriate wavelength of light and relax to the thermodynamically stable isomer in dark or with another wavelength of light. While photocaged PROTACs permit only activation control for protein degradation, photoswitching PROTACs offer reversible activation and deactivation by using suitable wavelengths of light.
    Keywords:  : protein degradation 1 * photocage 2 * azobenzene 3 *light 4 * UV 5
    DOI:  https://doi.org/10.1002/cmdc.202000249
  13. Mol Cell. 2020 Jun 18. pii: S1097-2765(20)30346-4. [Epub ahead of print]78(6): 991-993
    Liddicoat BJ, Dawson MA.
      Combination therapy remains the cornerstone for cancer management, and understanding how to rationally partner drugs is imperative. In this issue of Molecular Cell, Shu et al. (2020) provide a tour de force multi-omic approach to identify synergistic pathways that increase the efficacy of BET bromodomain inhibitors in triple-negative breast cancer.
    DOI:  https://doi.org/10.1016/j.molcel.2020.05.026
  14. Mol Cell. 2020 Jun 18. pii: S1097-2765(20)30347-6. [Epub ahead of print]78(6): 996-998
    Olsen SN, Armstrong SA.
      In this month's issue of Cancer Cell, Su et al. (2020) describe two small-molecule inhibitors of the RNA demethylase FTO that demonstrate significant anti-tumor effects in various models of acute myeloid leukemia.
    DOI:  https://doi.org/10.1016/j.molcel.2020.05.027
  15. J Biol Chem. 2020 Jun 18. pii: jbc.RA120.014259. [Epub ahead of print]
    Bibo-Verdugo B, Snipas SJ, Kolt S, Poreba M, Salvesen GS.
      Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway signaled by caspase-1 and the noncanonical pathway regulated by mouse caspase-11 and human caspases-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors pro-interleukin-18 and pro-interleukin-1β (pro-IL18/pro-IL1β). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4'-the C-terminal gasdermin D region adjacent to the cleavage site-influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable to that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11-i.e. caspases-4 and -5-are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.
    Keywords:  Caspase-11; Gasdermin D; Inflammatory caspase; Innate immunity; Pyroptosis; caspase; cell death; cytokine; inflammation; interleukin 1 (IL-1); substrate specificity
    DOI:  https://doi.org/10.1074/jbc.RA120.014259
  16. Nat Commun. 2020 Jun 16. 11(1): 3045
    Ameur LB, Marie P, Thenoz M, Giraud G, Combe E, Claude JB, Lemaire S, Fontrodona N, Polveche H, Bastien M, Gessain A, Wattel E, Bourgeois CF, Auboeuf D, Mortreux F.
      Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB-responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.
    DOI:  https://doi.org/10.1038/s41467-020-16853-x
  17. Immunol Rev. 2020 Jun 20.
    Anand PK.
      Inflammasomes are multi-protein complexes that regulate the cleavage of cysteine protease caspase-1, secretion of inflammatory cytokines, and induction of inflammatory cell death, pyroptosis. Several members of the nod-like receptor family assemble inflammasome in response to specific ligands. An exception to this is the NLRP3 inflammasome which is activated by structurally diverse entities. Recent studies have suggested that NLRP3 might be a sensor of cellular homeostasis, and any perturbation in distinct metabolic pathways results in the activation of this inflammasome. Lipid metabolism is exceedingly important in maintaining cellular homeostasis, and it is recognized that cells and tissues undergo extensive lipid remodeling during activation and disease. Some lipids are involved in instigating chronic inflammatory diseases, and new studies have highlighted critical upstream roles for lipids, particularly cholesterol, in regulating inflammasome activation implying key functions for inflammasomes in diseases with defective lipid metabolism. The focus of this review is to highlight how lipids regulate inflammasome activation and how this leads to the progression of inflammatory diseases. The key roles of cholesterol metabolism in the activation of inflammasomes have been comprehensively discussed. Besides, the roles of oxysterols, fatty acids, phospholipids, and lipid second messengers are also summarized in the context of inflammasomes. The overriding theme is that lipid metabolism has numerous but complex functions in inflammasome activation. A detailed understanding of this area will help us develop therapeutic interventions for diseases where dysregulated lipid metabolism is the underlying cause.
    Keywords:  NLRP3; cholesterol; homeostasis; inflammasome; lipids; metabolism
    DOI:  https://doi.org/10.1111/imr.12891
  18. Semin Cell Dev Biol. 2020 Jun 15. pii: S1084-9521(19)30189-2. [Epub ahead of print]
    Leon K, Ott M.
      The Nonsense-mediated mRNA Decay (NMD) pathway is an RNA quality control pathway conserved among eukaryotic cells. While historically thought to predominantly recognize transcripts with premature termination codons, it is now known that the NMD pathway plays a variety of roles, from homeostatic events to control of viral pathogens. In this review we highlight the reciprocal interactions between the host NMD pathway and viral pathogens, which have shaped both the host antiviral defense and viral pathogenesis.
    Keywords:  Anti-viral response; Nonsense-mediated mRNA decay; RNA biology; Viruses
    DOI:  https://doi.org/10.1016/j.semcdb.2020.05.018
  19. Nat Commun. 2020 Jun 15. 11(1): 3013
    Ng AP, Coughlan HD, Hediyeh-Zadeh S, Behrens K, Johanson TM, Low MSY, Bell CC, Gilan O, Chan YC, Kueh AJ, Boudier T, Feltham R, Gabrielyan A, DiRago L, Hyland CD, Ierino H, Mifsud S, Viney E, Willson T, Dawson MA, Allan RS, Herold MJ, Rogers K, Tarlinton DM, Smyth GK, Davis MJ, Nutt SL, Alexander WS.
      B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.
    DOI:  https://doi.org/10.1038/s41467-020-16828-y
  20. Int J Mol Sci. 2020 Jun 15. pii: E4266. [Epub ahead of print]21(12):
    Sarrou E, Richmond L, Carmody RJ, Gibson B, Keeshan K.
      Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.
    Keywords:  CRISPR/Cas9; chromosomal translocation; mixed lineage leukaemia; stem cells; tumourigenesis
    DOI:  https://doi.org/10.3390/ijms21124266
  21. Genome Biol. 2020 Jun 15. 21(1): 140
    Kerr CH, Skinnider MA, Andrews DDT, Madero AM, Chan QWT, Stacey RG, Stoynov N, Jan E, Foster LJ.
      BACKGROUND: The type I interferon (IFN) response is an ancient pathway that protects cells against viral pathogens by inducing the transcription of hundreds of IFN-stimulated genes. Comprehensive catalogs of IFN-stimulated genes have been established across species and cell types by transcriptomic and biochemical approaches, but their antiviral mechanisms remain incompletely characterized. Here, we apply a combination of quantitative proteomic approaches to describe the effects of IFN signaling on the human proteome, and apply protein correlation profiling to map IFN-induced rearrangements in the human protein-protein interaction network.RESULTS: We identify > 26,000 protein interactions in IFN-stimulated and unstimulated cells, many of which involve proteins associated with human disease and are observed exclusively within the IFN-stimulated network. Differential network analysis reveals interaction rewiring across a surprisingly broad spectrum of cellular pathways in the antiviral response. We identify IFN-dependent protein-protein interactions mediating novel regulatory mechanisms at the transcriptional and translational levels, with one such interaction modulating the transcriptional activity of STAT1. Moreover, we reveal IFN-dependent changes in ribosomal composition that act to buffer IFN-stimulated gene protein synthesis.
    CONCLUSIONS: Our map of the IFN interactome provides a global view of the complex cellular networks activated during the antiviral response, placing IFN-stimulated genes in a functional context, and serves as a framework to understand how these networks are dysregulated in autoimmune or inflammatory disease.
    Keywords:  Innate immunity; Interactome; Interferon; Interferon-stimulated gene; Protein complexes; Protein correlation profiling; Proteomics
    DOI:  https://doi.org/10.1186/s13059-020-02050-y
  22. Nucleic Acids Res. 2020 Jun 15. pii: gkaa508. [Epub ahead of print]
    Dhoonmoon A, Schleicher EM, Clements KE, Nicolae CM, Moldovan GL.
      The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.
    DOI:  https://doi.org/10.1093/nar/gkaa508
  23. J Allergy Clin Immunol. 2020 Jun 16. pii: S0091-6749(20)30630-8. [Epub ahead of print]
    Schön MP, Manzke V, Erpenbeck L.
      Research into the pathophysiology of psoriasis remains challenging, because this disease does not occur naturally in laboratory animals. However, specific aspects of its complex immune-pathology can be illuminated through transgenic, knockout, xenotransplantation, immunological reconstitution, drug-induced, or spontaneous mutation models in rodents. Although some of these approaches have already been pursued for more than 5 decades and even more models have been described in recent times, they have surprisingly not yet been systematically validated. As a consequence, researchers regularly examine specific aspects that only partially reflect the complex overall picture of the human disease. Nonetheless, animal models are of great utility to investigate inflammatory mediators, the communication between cells of the innate and the adaptive immune systems, the role of resident cells as well as new therapies. Of note, various manipulations in experimental animals resulted in rather similar phenotypes. These were called "psoriasiform", "psoriasis-like" or even "psoriasis" usually on the basis of some similarities with the human disorder. Xenotransplantation of human skin onto immunocompromised animals can overcome this limitation only in part. In this review, we elucidate approaches for the generation of animal models of psoriasis and assess their strengths and limitations with a certain focus on more recently developed models.
    Keywords:  Psoriasis; adoptive transfer; animal model; chronic inflammation; dermatology; drug testing; epidermis; hyperproliferation; imiquimod; preclinical; preclinical therapeutic study; skin immunity; skin inflammation; transgenic; xenotransplantation
    DOI:  https://doi.org/10.1016/j.jaci.2020.04.034
  24. Autoimmun Rev. 2020 Jun 16. pii: S1568-9972(20)30154-3. [Epub ahead of print] 102590
    Triggianese P, Novelli L, Galdiero MR, Chimenti MS, Conigliaro P, Perricone R, Perricone C, Gerli R.
      OBJECTIVE: To evaluate prevalence and clinical features of immune-related adverse events (irAEs) to immune checkpoint inhibitors (ICIs) in accordance with the gender of treated cancer patients.METHODS: A systematic review of the medical literature was conducted by searching all available clinical data up to December 2019 in several databases using a combination of MESH terms related to immune checkpoint inhibitors, autoimmunity, and gender. Analyzed data were related to all FDA approved ICIs and respective indications in cancer.
    RESULTS: According to data from the literature, male display a slightly lower frequencies of ICIs-related endocrinopathies compared with females, specifically thyroid dysfunction. On the contrary, ICIs-hypophysitis has been reported at higher rates among males compared with females. ICI-induced Sicca/Sjogren's syndrome showed a more frequent occurrence in men than the idiopathic primary form. No differences in gender distribution seem to arise in hematologic and gastrointestinal-irAEs. Interestingly, the gender distribution of neurologic and vascular ICIs-irAEs appears male-dominant.
    CONCLUSIONS: The present systematic review highlights for the first time that the distribution of patients experiencing irAEs associated with ICIs changes among the genders according to the specific drug used, the frequency of the cancer and of the autoimmune conditions in the general population.
    Keywords:  Autoimmunity; Gender; Hormones; Immune checkpoint
    DOI:  https://doi.org/10.1016/j.autrev.2020.102590
  25. Autophagy. 2020 Jun 16.
    Chu Y, Kang Y, Yan C, Yang C, Zhang T, Huo H, Liu Y.
      Macroautophagy/autophagy is a membrane-mediated intracellular degradation pathway, through which bulky cytoplasmic content is digested in lysosomes. How the autophagy initiation and maturation steps are regulated is not clear. In this study, we found an E3 ubiquitin ligase complex, linear ubiquitin chain assembly complex (LUBAC) and a de-ubiquitinating enzyme (DUB) OTULIN localize to the phagophore area to control autophagy initiation and maturation. LUBAC key component RNF31/HOIP translocates to the LC3 puncta area when autophagy is induced. RNF31 knockdown inhibits autophagy initiation, and cells are more sensitive to bacterial infection. OTULIN knockdown, however, promotes autophagy initiation but blocks autophagy maturation. In OTULIN knockdown cells, excessive ubiquitinated ATG13 protein was recruited to the phagophore for prolonged expansion, and therefore inhibits autophagosome maturation. Together, our study provides evidence that LUBAC and OTULIN cooperatively regulate autophagy initiation and autophagosome maturation by mediating the linear ubiquitination and the stabilization of ATG13.
    Keywords:  ATG13; LUBAC; OTULIN; RNF31; autophagosome; autophagy; linear ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2020.1781393
  26. J Cell Sci. 2020 Jun 16. pii: jcs.240085. [Epub ahead of print]
    Schregle R, Mueller S, Legler DF, Rossy J, Krueger WA, Groettrup M.
      Dendritic cell (DC) aggresome-like induced structures (DALIS) are protein aggregates of polyubiquitylated proteins that form transiently during DC maturation. DALIS scatter randomly throughout the cytosol and serve as antigen storage sites synchronising DC maturation and antigen presentation. Maturation of DCs is accompanied by the induction of the ubiquitin-like modifier FAT10 that localises to aggresomes that are structures similar to DALIS. FAT10 is conjugated to substrate proteins and serves as a signal for their rapid and irreversible degradation by the 26S proteasome similar to, yet independently of ubiquitin, thereby contributing to antigen presentation. Here we have investigated whether FAT10 is involved in the formation and turnover of DALIS and whether proteins accumulating in DALIS can be FAT10ylated. We found that FAT10 localises to DALIS in maturing DCs and that this localisation occurs independently of its conjugation to substrates. Additionally, we investigated the DALIS turnover in FAT10-deficient and -proficient DCs and observed FAT10-mediated disassembly of DALIS. Thus, we report further evidence that FAT10 is involved in antigen processing which may provide a functional rationale why FAT10 is selectively induced upon DC maturation.
    Keywords:  DALIS; Dendritic cells; FAT10; Proteasome; Ubiquitin
    DOI:  https://doi.org/10.1242/jcs.240085
  27. Immunity. 2020 Jun 14. pii: S1074-7613(20)30237-5. [Epub ahead of print]
    Liu Z, Wang C, Yang J, Chen Y, Zhou B, Abbott DW, Xiao TS.
      The recognition and cleavage of gasdermin D (GSDMD) by inflammatory caspases-1, 4, 5, and 11 are essential steps in initiating pyroptosis after inflammasome activation. Previous work has identified cleavage site signatures in substrates such as GSDMD, but it is unclear whether these are the sole determinants for caspase engagement. Here we report the crystal structure of a complex between human caspase-1 and the full-length murine GSDMD. In addition to engagement of the GSDMD N- and C-domain linker by the caspase-1 active site, an anti-parallel β sheet at the caspase-1 L2 and L2' loops bound a hydrophobic pocket within the GSDMD C-terminal domain distal to its N-terminal domain. This "exosite" interface endows an additional function for the GSDMD C-terminal domain as a caspase-recruitment module besides its role in autoinhibition. Our study thus reveals dual-interface engagement of GSDMD by caspase-1, which may be applicable to other physiological substrates of caspases.
    Keywords:  active site; crystal structure; dual site engagement; exosite; gasdermin D; inflammasome; inflammatory caspases; pyroptosis
    DOI:  https://doi.org/10.1016/j.immuni.2020.06.007