bims-tunefa Biomed News
on Tumor necrosis factor superfamily and post-translational modifications
Issue of 2020‒03‒29
thirty-six papers selected by
John Silke
Walter and Eliza Hall Institute of Medical Research


  1. Cell Mol Immunol. 2020 Mar 19.
    Tartey S, Gurung P, Karki R, Burton A, Hertzog P, Kanneganti TD.
      The SHP-1 protein encoded by the Ptpn6 gene has been extensively studied in hematopoietic cells in the context of inflammation. A point mutation in this gene (Ptpn6spin) causes spontaneous inflammation in mice, which has a striking similarity to neutrophilic dermatoses in humans. Recent findings highlighted the role of signaling adapters and kinases in promoting inflammation in Ptpn6spin mice; however, the underlying transcriptional regulation is poorly understood. Here, we report that SYK is important for driving neutrophil infiltration and initiating wound healing responses in Ptpn6spin mice. Moreover, we found that deletion of the transcription factor Ets2 in myeloid cells ameliorates cutaneous inflammatory disease in Ptpn6spin mice through transcriptional regulation of its target inflammatory genes. Furthermore, Ets-2 drives IL-1α-mediated inflammatory signaling in neutrophils of Ptpn6spin mice. Overall, in addition to its well-known role in driving inflammation in cancer, Ets-2 plays a major role in regulating IL-1α-driven Ptpn6spin-mediated neutrophilic dermatoses. Model for the role of ETS-2 in neutrophilic inflammation in Ptpn6spin mice. Mutation of the Ptpn6 gene results in SYK phosphorylation which then sequentially activates MAPK signaling pathways and activation of ETS-2. This leads to activation of ETS-2 target genes that contribute to neutrophil migration and inflammation. When Ets2 is deleted in Ptpn6spin mice, the expression of these target genes is reduced, leading to the reduced pathology in neutrophilic dermatoses.
    Keywords:  Autoinflammation; ETS-2; IL-1α; Neutrophilic dermatoses; PTPN6; SHP-1
    DOI:  https://doi.org/10.1038/s41423-020-0398-7
  2. Nat Immunol. 2020 Apr;21(4): 381-387
    Martens A, Priem D, Hoste E, Vetters J, Rennen S, Catrysse L, Voet S, Deelen L, Sze M, Vikkula H, Slowicka K, Hochepied T, Iliaki K, Wullaert A, Janssens S, Lamkanfi M, Beyaert R, Armaka M, Bertrand MJM, van Loo G.
      Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties.
    DOI:  https://doi.org/10.1038/s41590-020-0621-9
  3. Leukemia. 2020 Mar 16.
    Best S, Lam V, Liu T, Bruss N, Kittai A, Danilova OV, Murray S, Berger A, Pennock ND, Lind EF, Danilov AV.
      Novel targeted agents used in therapy of lymphoid malignancies, such as inhibitors of B-cell receptor-associated kinases, are recognized to have complex immune-mediated effects. NEDD8-activating enzyme (NAE) has been identified as a tractable target in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma. We and others have shown that pevonedistat (TAK-924), a small-molecule inhibitor of NAE, abrogates NF-κB signaling in malignant B cells. However, NF-κB pathway activity is indispensable in immune response, and T-cell function is altered in patients with CLL. Using T cells derived from patients with CLL, we demonstrate that although targeting NAE results in markedly differential expression of NF-κB-regulated genes and downregulation of interleukin (IL)-2 signaling during T-cell activation, T cells evade apoptosis. Meanwhile, NAE inhibition favorably modulates polarization of T cells in vitro, with decreased Treg differentiation and a shift toward TH1 phenotype, accompanied by increased interferon-γ production. These findings were recapitulated in vivo in immunocompetent mouse models. T cells exposed to pevonedistat in washout experiments, informed by its human pharmacokinetic profile, recover NAE activity, and maintain their response to T-cell receptor stimulation and cytotoxic potential. Our data shed light on the potential immune implications of targeting neddylation in CLL and lymphoid malignancies.
    DOI:  https://doi.org/10.1038/s41375-020-0794-0
  4. Semin Cancer Biol. 2020 Mar 18. pii: S1044-579X(20)30065-1. [Epub ahead of print]
    Nguyen KM, Busino L.
      Small molecule-mediated inhibition of protein function is the rational behind therapeutic efficacy of the majority clinically used drugs. In order for a drug to achieve pharmacologically relevant inhibition, efficient target engagement at high selectivity and specificity is necessary to obtain the desired therapeutic effect minimizing offtarget outcomes. Majority of small molecules approaches developed so far have failed in their attempt to reach clinical efficacy because of low selectivity and low specificity in achieving close to 100% target inhibition. Recently, approaches that directly control cellular protein levels have opened the potential to accomplish a high grade of efficacy not imaginable with traditional small-molecule inhibitors. Research in this area has just started opening avenues to effectively degrade a cellular target of choice and will soon impact clinical efficacy.
    Keywords:  CRBN; Cereblon; Cullin-Ring ligasem lymphoma; DCAF15; PROTAC; molecular glue; myeloma; ubiquitin
    DOI:  https://doi.org/10.1016/j.semcancer.2020.03.007
  5. Mol Cell Biol. 2020 Mar 23. pii: MCB.00040-20. [Epub ahead of print]
    O'Brien ME, Londino J, McGinnis M, Weathington N, Adair J, Suber T, Kagan V, Chen K, Zou C, Chen B, Bon J, Mallampalli RK.
      FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here we show that TNFα, a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation as reflected by reduced expression of myogenin and impaired myotube formation. TNFα did not destabilize the Fbxl2 transcript (t ½ ∼10 h), but inhibited SP1 transactivation of its core promoter, localized to 160/+42 base pairs within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect less pronounced during proliferation or after TNFα exposure. TNFα, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key pro-inflammatory myopathic cytokine.Importance Skeletal muscle regeneration and repair involves the recruitment and proliferation of resident satellite cells that exit the cell cycle during the process of myogenic differentiation to form myofibers. We demonstrate that the ubiquitin E3 ligase subunit FBXL2 is essential for skeletal myogenesis through its important effects on cell cycle progression and cell proliferative signaling. Further, we characterize a new mechanism whereby sustained stimulation by a major pro-inflammatory cytokine, TNFα, regulates skeletal myogenesis by inhibiting the interaction of SP1 with the Fbxl2 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of Fbxl2 as both a critical regulator of myogenic proliferative processes and a susceptible gene target during inflammatory stimulation by TNFα in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained pro-inflammatory signaling.
    DOI:  https://doi.org/10.1128/MCB.00040-20
  6. J Immunol. 2020 Mar 23. pii: ji1901508. [Epub ahead of print]
    Karki R, Lee E, Sharma BR, Banoth B, Kanneganti TD.
      Inflammasomes are intracellular signaling complexes that are assembled in response to a variety of pathogenic or physiologic stimuli to initiate inflammatory responses. Ubiquitously present LPS in Gram-negative bacteria induces NLRP3 inflammasome activation that requires caspase-11. We have recently demonstrated that IFN regulatory factor (IRF) 8 was dispensable for caspase-11-mediated NLRP3 inflammasome activation during LPS transfection; however, its role in Gram-negative bacteria-mediated NLRP3 inflammasome activation remains unknown. In this study, we found that IRF8 promotes NLRP3 inflammasome activation in murine bone marrow-derived macrophages (BMDMs) infected with Gram-negative bacteria such as Citrobacter rodentium, Escherichia coli, or Pseudomonas aeruginosa mutant strain ΔpopB Moreover, BMDMs deficient in IRF8 showed substantially reduced caspase-11 activation and gasdermin D cleavage, which are required for NLRP3 inflammasome activation. Mechanistically, IRF8-mediated phosphorylation of IRF3 was required for Ifnb transcription, which in turn triggered the caspase-11-dependent NLRP3 inflammasome activation in the infected BMDMs. Overall, our findings suggest that IRF8 promotes NLRP3 inflammasome activation during infection with Gram-negative bacteria.
    DOI:  https://doi.org/10.4049/jimmunol.1901508
  7. FASEB J. 2020 Mar 22.
    Seabright AP, Fine NHF, Barlow JP, Lord SO, Musa I, Gray A, Bryant JA, Banzhaf M, Lavery GG, Hardie DG, Hodson DJ, Philp A, Lai YC.
      Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.
    Keywords:  endogenous; mitophagy; skeletal muscle; tandem ubiquitin-binding entity (TUBE); ubiquitin
    DOI:  https://doi.org/10.1096/fj.201903051R
  8. Cell Mol Immunol. 2020 Mar 19.
    Zhou X, Xie F, Wang L, Zhang L, Zhang S, Fang M, Zhou F.
      The innate immune system plays a crucial role in the host defense against viral and microbial infection. Exosomes constitute a subset of extracellular vesicles (EVs) that can be released by almost all cell types. Owing to their capacity to shield the payload from degradation and to evade recognition and subsequent removal by the immune system, exosomes efficiently transport functional components to recipient cells. Accumulating evidence has recently shown that exosomes derived from tumor cells, host cells and even bacteria and parasites mediate the communication between the invader and innate immune cells and thus play an irreplaceable function in the dissemination of pathogens and donor cell-derived molecules, modulating the innate immune responses of the host. In this review, we describe the current understanding of EVs (mainly focusing on exosomes) and summarize and discuss their crucial roles in determining innate immune responses. Additionally, we discuss the potential of using exosomes as biomarkers and cancer vaccines in diagnostic and therapeutic applications.
    Keywords:  cancer diagnosis; exosome; extracellular vesicles; immunotherapy; innate immune
    DOI:  https://doi.org/10.1038/s41423-020-0391-1
  9. Cytokine. 2020 Mar 21. pii: S1043-4666(20)30082-X. [Epub ahead of print]130 155066
    Mahdavi Sharif P, Jabbari P, Razi S, Keshavarz-Fathi M, Rezaei N.
      TNF-alpha is involved in many physiologic and pathologic cellular pathways, including cellular proliferation, differentiation, and death, regulation of immunologic reactions to different cells and molecules, local and vascular invasion of neoplasms, and destruction of tumor vasculature. It is obvious that because of integrated functions of TNF-alpha inside different physiologic systems, it cannot be used as a single-agent therapy for neoplasms; however, long-term investigation of its different cellular pathways has led to recognition of a variety of subsequent molecules with more specific interactions, and therefore, might be suitable as prognostic and therapeutic factors for neoplasms. Here, we will review different aspects of the TNF-alpha as a cytokine involved in both physiologic functions of cells and pathologic abnormalities, most importantly, cancers.
    Keywords:  Angiogenesis; Matrix metalloproteinase; Neoplasm; Nuclear factor kappa B; Tumor necrosis factor-alpha
    DOI:  https://doi.org/10.1016/j.cyto.2020.155066
  10. EMBO Rep. 2020 Mar 23. e49799
    Riley JS, Tait SW.
      Mitochondria are cellular organelles that orchestrate a vast range of biological processes, from energy production and metabolism to cell death and inflammation. Despite this seemingly symbiotic relationship, mitochondria harbour within them a potent agonist of innate immunity: their own genome. Release of mitochondrial DNA into the cytoplasm and out into the extracellular milieu activates a plethora of different pattern recognition receptors and innate immune responses, including cGAS-STING, TLR9 and inflammasome formation leading to, among others, robust type I interferon responses. In this Review, we discuss how mtDNA can be released from the mitochondria, the various inflammatory pathways triggered by mtDNA release and its myriad biological consequences for health and disease.
    Keywords:  cell death; immunity; inflammation; mitochondria; mtDNA
    DOI:  https://doi.org/10.15252/embr.201949799
  11. Chem Commun (Camb). 2020 Mar 23.
    Smalley JP, Adams GE, Millard CJ, Song Y, Norris JKS, Schwabe JWR, Cowley SM, Hodgkinson JT.
      We have identified a proteolysis targeting chimera (PROTAC) of class I HDACs 1, 2 and 3. The most active degrader consists of a benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-Lindau E3 ligand. Our PROTAC increased histone acetylation levels and compromised colon cancer HCT116 cell viability, establishing a degradation strategy as an alternative to class I HDAC inhibition.
    DOI:  https://doi.org/10.1039/d0cc01485k
  12. Nature. 2020 Mar;579(7800): 592-597
    Gallego LD, Schneider M, Mittal C, Romanauska A, Gudino Carrillo RM, Schubert T, Pugh BF, Köhler A.
      The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle1. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.
    DOI:  https://doi.org/10.1038/s41586-020-2097-z
  13. Blood Rev. 2020 Feb 21. pii: S0268-960X(20)30022-9. [Epub ahead of print] 100672
    Wei AH, Roberts AW, Spencer A, Rosenberg AS, Siegel D, Walter RB, Caenepeel S, Hughes P, McIver Z, Mezzi K, Morrow PK, Stein A.
      Myeloid cell leukemia sequence 1 (MCL-1) is an antiapoptotic protein that plays a key role in promoting cell survival in multiple myeloma (MM), acute myeloid leukemia (AML), and non-Hodgkin lymphoma (NHL). Overexpression of MCL-1 is associated with treatment resistance and poor prognosis; thus, MCL-1 inhibitors are rational therapeutic options for malignancies depending on MCL-1. Several MCL-1 inhibitors have entered clinical trials, including AZD5991, S64315, AMG 176, and AMG 397. A key area of investigation is whether MCL-1 inhibitors will complement the activity of BCL-2 inhibitors, such as venetoclax, and synergistically enhance anti-tumor efficacy when given in combination with other anti-cancer drugs. Another important question is whether a safe therapeutic window can be found for this new class of inhibitors. In summary, inhibition of MCL-1 shows potential as a treatment for hematologic malignancies and clinical evaluation of MCL-1 inhibitors is currently underway.
    Keywords:  Acute myeloid leukemia; BH3-mimetic; MCL-1 inhibitor; Multiple myeloma; Non-Hodgkin lymphoma
    DOI:  https://doi.org/10.1016/j.blre.2020.100672
  14. J Immunol. 2020 Mar 25. pii: ji1900281. [Epub ahead of print]
    Alfano DN, Klei LR, Klei HB, Trotta M, Gough PJ, Foley KP, Bertin J, Sumpter TL, Lucas PC, McAllister-Lucas LM.
      The signaling protein MALT1 plays a key role in promoting NF-κB activation in Ag-stimulated lymphocytes. In this capacity, MALT1 has two functions, acting as a scaffolding protein and as a substrate-specific protease. MALT1 is also required for NF-κB-dependent induction of proinflammatory cytokines after FcεR1 stimulation in mast cells, implicating a role in allergy. Because MALT1 remains understudied in this context, we sought to investigate how MALT1 proteolytic activity contributes to the overall allergic response. We compared bone marrow-derived mast cells from MALT1 knockout (MALT1-/-) and MALT1 protease-deficient (MALTPD/PD) mice to wild-type cells. We found that MALT1-/- and MALT1PD/PD mast cells are equally impaired in cytokine production following FcεRI stimulation, indicating that MALT1 scaffolding activity is insufficient to drive the cytokine response and that MALT1 protease activity is essential. In addition to cytokine production, acute mast cell degranulation is a critical component of allergic response. Intriguingly, whereas degranulation is MALT1-independent, MALT1PD/PD mice are protected from vascular edema induced by either passive cutaneous anaphylaxis or direct challenge with histamine, a major granule component. This suggests a role for MALT1 protease activity in endothelial cells targeted by mast cell-derived vasoactive substances. Indeed, we find that in human endothelial cells, MALT1 protease is activated following histamine treatment and is required for histamine-induced permeability. We thus propose a dual role for MALT1 protease in allergic response, mediating 1) IgE-dependent mast cell cytokine production, and 2) histamine-induced endothelial permeability. This dual role indicates that therapeutic inhibitors of MALT1 protease could work synergistically to control IgE-mediated allergic disease.
    DOI:  https://doi.org/10.4049/jimmunol.1900281
  15. Front Neurosci. 2020 ;14 193
    Ahmadi Rastegar D, Dzamko N.
      For more than a decade, researchers have sought to uncover the biological function of the enigmatic leucine rich repeat kinase 2 (LRRK2) enzyme, a large multi-domain protein with dual GTPase and kinase activities. Originally identified as a familial Parkinson's disease (PD) risk gene, variations in LRRK2 are also associated with risk of idiopathic PD, inflammatory bowel disease and susceptibility to bacterial infections. LRRK2 is highly expressed in peripheral immune cells and the potential of LRRK2 to regulate immune and inflammatory pathways has emerged as common link across LRRK2-implicated diseases. This review outlines the current genetic and biochemical evidence linking LRRK2 to the regulation of innate immune inflammatory pathways, including the toll-like receptor and inflammasome pathways. Evidence suggests a complex interplay between genetic risk and protective alleles acts to modulate immune outcomes in a manner dependent on the particular pathogen and cell type invaded.
    Keywords:  Crohn’s; LRRK2; Parkinson’s; inflammasome; inflammation; monocyte; toll-like receptor
    DOI:  https://doi.org/10.3389/fnins.2020.00193
  16. Semin Cancer Biol. 2020 Mar 19. pii: S1044-579X(20)30066-3. [Epub ahead of print]
    Bonacci T, Emanuele MJ.
      Since its discovery forty years ago, protein ubiquitination has been an ever-expanding field. Virtually all biological processes are controlled by the post-translational conjugation of ubiquitin onto target proteins. In addition, since ubiquitin controls substrate degradation through the action of hundreds of enzymes, many of which represent attractive therapeutic candidates, harnessing the ubiquitin system to reshape proteomes holds great promise for improving disease outcomes. Among the numerous physiological functions controlled by ubiquitin, the cell cycle is among the most critical. Indeed, the discovery that the key drivers of cell cycle progression are regulated by the ubiquitin-proteasome system (UPS) epitomizes the connection between ubiquitin signaling and proliferation. Since cancer is a disease of uncontrolled cell cycle progression and proliferation, targeting the UPS to stop cancer cells from cycling and proliferating holds enormous therapeutic potential. Ubiquitination is reversible, and ubiquitin is removed from substrates by catalytic proteases termed deubiquitinases or DUBs. While ubiquitination is tightly linked to proliferation and cancer, the role of DUBs represents a layer of complexity in this landscape that remains poorly captured. Due to their ability to remodel the proteome by altering protein degradation dynamics, DUBs play an important and underappreciated role in the cell cycle and proliferation of both normal and cancer cells. Moreover, due to their enzymatic protease activity and an open ubiquitin binding pocket, DUBs are likely to be important in the future of cancer treatment, since they are among the most druggable enzymes in the UPS. In this review, we summarize new and important findings linking DUBs to cell cycle and proliferation, as well as to the etiology and treatment of cancer. We also highlight new advances in developing pharmacological approaches to attack DUBs for therapeutic benefit.
    Keywords:  Cell cycle; DUB; bap1; cezanne; cyld; deubiquitinases; otud7b; usp2; usp21; usp22; usp35; usp37; usp44; usp7; usp9x
    DOI:  https://doi.org/10.1016/j.semcancer.2020.03.008
  17. J Bone Miner Res. 2020 Mar 25.
    Li Q, Wang M, Xue H, Liu W, Guo Y, Xu R, Shao B, Yuan Q.
      The ubiquitination and deubiquitination enzymes ensure the stability and proper function of most cellular proteins. Disturbance of either enzyme compromises tissue homeostasis. We recently have identified that the ubiquitin-specific protease 34 (USP34) contributes to bone formation by promoting osteogenic differentiation of mesenchymal stem cells. However, its role in bone resorption, which couples bone formation, remains unknown. Here we show knockdown of Usp34 promotes osteoclast differentiation of RAW264.7 cells. Conditional knockout of Usp34 in bone marrow-derived macrophages (BMMs) or in osteoclasts leads to elevated osteoclast function and low bone mass. Mechanically, we identify that USP34 restrains NF-κB signaling by deubiquitinating and stabilizing the NF-κB inhibitor alpha (IκBα). Overexpression of IκBα represses osteoclastic hyperfunction of Usp34-deficient RAW264.7 cells. Collectively, our results demonstrate that USP34 inhibits osteoclastogenesis by regulating NF-κB signaling. This article is protected by copyright. All rights reserved.
    Keywords:  Molecular pathways - remodeling; Osteoclasts; Osteopenia
    DOI:  https://doi.org/10.1002/jbmr.4015
  18. Bioorg Med Chem Lett. 2020 Mar 10. pii: S0960-894X(20)30193-1. [Epub ahead of print] 127104
    Dorich S, Cox JH, Burch JD, Chagnon F, Chen B, Léger S, St-Onge M, Fader LD.
      Novel prostaglandin E2 receptor 4 (EP4) agonists featuring a pyridone core and an allylic alcohol ω-chain were discovered. These agonists were shown to be selective over EP1, EP2 and EP3. Analogs harboring a 4-carboxylic acid phenethyl α-chain displayed improved potency over those containing an n-heptanoic acid chain. Key SAR relationships were also identified.
    Keywords:  Agonists; Allylic alcohol; Prostaglandin E2 receptor 4 (EP(4)); Pyridone
    DOI:  https://doi.org/10.1016/j.bmcl.2020.127104
  19. Cell Mol Life Sci. 2020 Mar 21.
    Pascoe AL, Johnston AJ, Murphy RM.
      Skeletal muscle is one of the largest functional tissues in the human body; it is highly plastic and responds dramatically to anabolic and catabolic stimuli, including weight training and malnutrition, respectively. Excessive loss of muscle mass, or atrophy, is a common symptom of many disease states with severe impacts on prognosis and quality of life. TNF-like weak inducer of apoptosis (TWEAK) and its cognate receptor, fibroblast growth factor-inducible 14 (Fn14) are an emerging cytokine signaling pathway in the pathogenesis of muscle atrophy. Upregulation of TWEAK and Fn14 has been described in a number of atrophic and injured muscle states; however, it remains unclear whether they are contributing to the degenerative or regenerative aspect of muscle insults. The current review focuses on the expression and apparent downstream outcomes of both TWEAK and Fn14 in a range of catabolic and anabolic muscle models. Apparent changes in the signaling outcomes of TWEAK-Fn14 activation dependent on the relative expression of both the ligand and the receptor are discussed as a potential source of divergent TWEAK-Fn14 downstream effects. This review proposes both a physiological and pathological model of TWEAK-Fn14 signaling. Further research is needed on the switch between these states to develop therapeutic interventions for this pathway.
    Keywords:  Cachexia; Differentiation; Muscle loss; Myogenesis; NFκb; Proliferation
    DOI:  https://doi.org/10.1007/s00018-020-03495-x
  20. Am J Ophthalmol. 2020 Mar 21. pii: S0002-9394(20)30113-6. [Epub ahead of print]
    Chen H, Chen H, Liang L, Zhong Y, Liang Y, Yu Y, Huang S, Lu X.
      PURPOSE: To compare tear protein markers between normal subjects and dry eye (DE) patients with high and low lymphotoxin (LT)-alpha levels.DESIGN: Prospective cross-sectional study.
    METHODS: DE patients were divided into low (≤700 pg/ml) and high (>700 pg/ml) LT-alpha groups. Twelve protein markers were measured by microsphere-based immunoassay and ocular surface parameters were determined in right eyes (33 high LT-alpha DE, 27 low LT-alpha DE, 20 control) and left eyes (21 high LT-alpha DE, 39 low-LT-alpha DE, 20 control).
    RESULTS: In both eyes, tumor necrosis factor-α (TNF-α), interleukin (IL)-10, IL-1beta, IL-1Ra, IL-17A, and IL-12/23 p40 levels in high LT-alpha DE were significantly higher (p<0.01) than in low LT-alpha DE. Significant correlations identified in high LT-alpha DE were: SPEED with IL-10 (R=0.43, P=0.013), IL-1beta (R=0.4 8, P=0.005), and IL-12/23 p40 (R=0.50, P=0.003); IL-12/23 p40 with ocular surface disease index (OSDI) (R=0.35, P =0.049); and epidermal growth factor (EGF) with corneal fluorescein staining (CFS) score (R=-0.36, P=0.038). Significant correlations in low LT-alpha DE were: SPEED with IL-10 (R=-0.39, P=0.046), TNF-α (R=-0.39, P=0.047), and IL-17A (R=-0.48, P=0.013); OSDI with TNF-α (R=-0.47, P=0.017) and IL-17A (R=-0.46, P=0.018); and IL-6 with tear breakup time (R=-0.40, P=0.044). Lastly, IL-1Ra levels significantly increased in DE patients, positively correlated with temporal conjunctival hyperemia index (TCHI) and negatively correlated with Schirmer I test (p<0.05).
    CONCLUSIONS: Our study identified tear IL-1Ra level as a potential biomarker to replace Schirmer I test. Multiple tear protein marker levels increased in high LT-alpha DE, indicating that high LT-alpha DE might have a different pathogenesis.
    Keywords:  Dry Eye; LT-alpha; Pathogenesis; Tears Protein Markers
    DOI:  https://doi.org/10.1016/j.ajo.2020.03.013
  21. Cancer Res. 2020 Mar 25. pii: canres.3033.2019. [Epub ahead of print]
    Krais JJ, Wang Y, Bernhardy AJ, Clausen E, Miller JA, Cai KQ, Scott CL, Johnson N.
      BRCA1 gene mutations impair homologous recombination (HR) DNA repair, resulting in cellular senescence and embryonic lethality in mice. Therefore, BRCA1-deficient cancers require adaptations that prevent excessive genomic alterations from triggering cell death. RNF168-mediated ubiquitination of γH2AX at K13/15 (ub-H2AX) serves as a recruitment module for the localization of 53BP1 to DNA break sites. Here, we found multiple BRCA1 mutant cancer cell lines and primary tumors with low levels of RNF168 protein expression. Overexpression of ectopic RNF168 or a ub-H2AX fusion protein induced cell death and delayed BRCA1 mutant tumor formation. Cell death resulted from the recruitment of 53BP1 to DNA break sites and inhibition of DNA end resection. Strikingly, re-introduction of BRCA1 or 53BP1 depletion restored HR and rescued the ability of cells to maintain RNF168 and ub-H2AX overexpression. Thus, downregulation of RNF168 protein expression is a mechanism for providing BRCA1 null cancer cell lines with a residual level of HR that is essential for viability. Overall, our work identifies loss of RNF168 ubiquitin signaling as a proteomic alteration that supports BRCA1 mutant carcinogenesis. We propose that restoring RNF168-ub-H2AX signaling, potentially through inhibition of de-ubiquitinases, could represent a new therapeutic approach.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-3033
  22. Nat Immunol. 2020 Apr;21(4): 422-433
    Razani B, Whang MI, Kim FS, Nakamura MC, Sun X, Advincula R, Turnbaugh JA, Pendse M, Tanbun P, Achacoso P, Turnbaugh PJ, Malynn BA, Ma A.
      A20 is an anti-inflammatory protein that is strongly linked to human disease. Here, we find that mice expressing three distinct targeted mutations of A20's zinc finger 7 (ZF7) ubiquitin-binding motif uniformly developed digit arthritis with features common to psoriatic arthritis, while mice expressing point mutations in A20's OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20ZF7 cells exhibited prolonged IκB kinase activity that drove exaggerated transcription of late-phase nuclear factor-κB response genes in vitro and in prediseased mouse paws in vivo. In addition, mice expressing double-mutant A20 proteins in A20's ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20's ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.
    DOI:  https://doi.org/10.1038/s41590-020-0634-4
  23. J Med Chem. 2020 Mar 27.
    Li Z, Li X, Su MB, Gao LX, Zhou YB, Yuan B, Lyu X, Yan Z, Hu C, Zhang H, Luo C, Chen Z, Li J, Zhao Y.
      The overexpression of NIK plays a critical role in liver inflammatory diseases. Treatment of such diseases with small-molecule NIK inhibitors is a reasonable but underexplored approach. In this paper, we reported the discovery of a potent and selective NIK inhibitor 46 (XT2). 46 inhibited the NIK kinase with an IC50 value of 9.1 nM in vitro, and it also potently suppressed NIK activities in intact cells. In isogenic primary hepatocytes, treatment of 46 efficiently suppressed the expressions of NIK-induced gene. 46 was orally bioavailable in mice with moderate systemic exposure. In a NIK-associated mouse liver inflammation model, 46 suppressed CCl4-induced upregulation of ALT, a key biomarker of acute liver injury. 46 also decreased immune cell infiltration into the injured liver tissue. Overall, these studies provide examples that an NIK inhibitor is able to suppress toxin-induced liver inflammations, which indicates its therapeutic potentials for the treatment of liver inflammatory diseases.
    DOI:  https://doi.org/10.1021/acs.jmedchem.0c00396
  24. Genes Dis. 2020 Jun;7(2): 166-171
    Sreedhar A, Wiese EK, Hitosugi T.
      Lysine succinylation (Ksucc), defined as a transfer of a succinyl group to a lysine residue of a protein, is a newly identified protein post-translational modification1-3. This chemical modification is reversible, dynamic, and evolutionarily conserved 4 where it has been comprehensively studied in both bacterial and mammalian cells5-7. Numerous proteins involved in the regulation of various cellular and biological processes have been shown to be heavily succinylated5-7. Emerging clinical data provides evidence that dysregulation of Ksucc is correlated with the development of several diseases, including cardiovascular diseases and cancer7-9. Therefore, an in-depth understanding of Ksucc and its regulation is important not only for understanding its physiological function but also for developing drug therapies and targeted agents for these diseases. In this review, we highlight some of the recent advances in understanding the role of Ksucc and desuccinylation under physiological and pathological conditions.
    Keywords:  Lysine succinylation; Metabolism; Post-translational modification; SIRT5; Succinyl-CoA
    DOI:  https://doi.org/10.1016/j.gendis.2019.09.011
  25. Drugs R D. 2020 Mar 25.
    Boussios S, Abson C, Moschetta M, Rassy E, Karathanasi A, Bhat T, Ghumman F, Sheriff M, Pavlidis N.
      Genetic complexity and DNA damage repair defects are common in different cancer types and can induce tumor-specific vulnerabilities. Poly(ADP-ribose) polymerase (PARP) inhibitors exploit defects in the DNA repair pathway through synthetic lethality and have emerged as promising anticancer therapies, especially in tumors harboring deleterious germline or somatic breast cancer susceptibility gene (BRCA) mutations. However, the utility of PARP inhibitors could be expanded beyond germline BRCA1/2 mutated cancers by causing DNA damage with cytotoxic agents in the presence of a DNA repair inhibitor. US Food and Drug Administration (FDA)-approved PARP inhibitors include olaparib, rucaparib, and niraparib, while veliparib is in the late stage of clinical development. Talazoparib inhibits PARP catalytic activity, trapping PARP1/2 on damaged DNA, and it has been approved by the US FDA for the treatment of metastatic germline BRCA1/2 mutated breast cancers in October 2018. The talazoparib side effect profile more closely resembles traditional chemotherapeutics rather than other clinically approved PARP inhibitors. In this review, we discuss the scientific evidence that has emerged from both experimental and clinical studies in the development of talazoparib. Future directions will include optimizing combination therapy with chemotherapy, immunotherapies and targeted therapies, and in developing and validating biomarkers for patient selection and stratification, particularly in malignancies with 'BRCAness'.
    DOI:  https://doi.org/10.1007/s40268-020-00301-8
  26. Trends Cell Biol. 2020 Apr;pii: S0962-8924(20)30018-0. [Epub ahead of print]30(4): 263-275
    Tiku V, Tan MW, Dikic I.
      Mitochondria have a central role in regulating a range of cellular activities and host responses upon bacterial infection. Multiple pathogens affect mitochondria dynamics and functions to influence their intracellular survival or evade host immunity. On the other side, major host responses elicited against infections are directly dependent on mitochondrial functions, thus placing mitochondria centrally in maintaining homeostasis upon infection. In this review, we summarize how different bacteria and viruses impact morphological and functional changes in host mitochondria and how this manipulation can influence microbial pathogenesis as well as the host cell metabolism and immune responses.
    Keywords:  bacteria; cell death; innate immunity; mitochondrial metabolism; mitochondrial morphology; viruses
    DOI:  https://doi.org/10.1016/j.tcb.2020.01.006
  27. J Immunol. 2020 Mar 25. pii: ji1900790. [Epub ahead of print]
    Bellamri N, Viel R, Morzadec C, Lecureur V, Joannes A, de Latour B, Llamas-Gutierrez F, Wollin L, Jouneau S, Vernhet L.
      The chemokine CXCL13 controls the normal organization of secondary lymphoid tissues and the neogenesis of ectopic lymphoid structures in nonlymphoid organs, particularly the lungs. The progression and severity of idiopathic pulmonary fibrosis (IPF), a fatal and irreversible interstitial lung disease, is predicted by the circulating blood concentrations of CXCL13. Although CXCL13 is produced by pulmonary tissues, it has not been determined which cells are involved. This study examines CXCL13 production by lung tissue macrophages from patients with IPF and the signaling pathways controlling CXCL13 gene expression in human alveolar macrophages (AM) and monocyte-derived macrophages (MoDM). CXCL13 is found in CD68- and CD206-positive AM from patients with IPF, and the CXCL13 gene is induced in these macrophages and MoDM when they are stimulated with LPS. We found that TNF-α and IL-10 control optimal CXCL13 gene expression in MoDM and possibly in AM by activating the NF-κB and JAK/STAT pathways, respectively. We also found that blood TNF-α and CXCL13 concentrations are significantly correlated in patients with IPF, suggesting that TNF-α contributes to CXCL13 production in humans. In conclusion, the results of this study demonstrate that AM from patients with IPF produces CXCL13 and that the NF-κB and JAK/STAT pathways are required to induce the expression of this major chemokine.
    DOI:  https://doi.org/10.4049/jimmunol.1900790
  28. Cell Chem Biol. 2020 Mar 19. pii: S2451-9456(20)30070-2. [Epub ahead of print]27(3): 257-258
    Mayer G, Legen T, Patwari T, Weber AM.
      In a recent issue of Cell Chemical Biology, Gray et al. (2020) report an aptamer-based method to reversibly label and isolate EGF receptor-expressing cells from heterogeneous mixtures by cell sorting approaches. Subsequent treatment using complementary oligonucleotides restores full functionality of EGF receptors, highlighting the superiority of this method to antibody-based sorting.
    DOI:  https://doi.org/10.1016/j.chembiol.2020.02.006
  29. Arterioscler Thromb Vasc Biol. 2020 Mar 26. ATVBAHA119313640
    Rasheed A, Robichaud S, Nguyen MA, Geoffrion M, Wyatt H, Cottee ML, Dennison T, Pietrangelo A, Lee R, Lagace TA, Ouimet M, Rayner KJ.
      OBJECTIVES: During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase-like domain protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe-knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages.CONCLUSIONS: These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.
    Keywords:  atherosclerosis; cholesterol; foam cells; macrophages; necroptosis
    DOI:  https://doi.org/10.1161/ATVBAHA.119.313640
  30. Blood Adv. 2020 Mar 24. 4(6): 1145-1158
    Bellissimo DC, Chen CH, Zhu Q, Bagga S, Lee CT, He B, Wertheim GB, Jordan M, Tan K, Worthen GS, Gilliland DG, Speck NA.
      RUNX1 is frequently mutated in myeloid and lymphoid malignancies. It has been shown to negatively regulate Toll-like receptor 4 (TLR4) signaling through nuclear factor κB (NF-κB) in lung epithelial cells. Here we show that RUNX1 regulates TLR1/2 and TLR4 signaling and inflammatory cytokine production by neutrophils. Hematopoietic-specific RUNX1 loss increased the production of proinflammatory mediators, including tumor necrosis factor-α (TNF-α), by bone marrow neutrophils in response to TLR1/2 and TLR4 agonists. Hematopoietic RUNX1 loss also resulted in profound damage to the lung parenchyma following inhalation of the TLR4 ligand lipopolysaccharide (LPS). However, neutrophils with neutrophil-specific RUNX1 loss lacked the inflammatory phenotype caused by pan-hematopoietic RUNX1 loss, indicating that dysregulated TLR4 signaling is not due to loss of RUNX1 in neutrophils per se. Rather, single-cell RNA sequencing indicates the dysregulation originates in a neutrophil precursor. Enhanced inflammatory cytokine production by neutrophils following pan-hematopoietic RUNX1 loss correlated with increased degradation of the inhibitor of NF-κB signaling, and RUNX1-deficient neutrophils displayed broad transcriptional upregulation of many of the core components of the TLR4 signaling pathway. Hence, early, pan-hematopoietic RUNX1 loss de-represses an innate immune signaling transcriptional program that is maintained in terminally differentiated neutrophils, resulting in their hyperinflammatory state. We hypothesize that inflammatory cytokine production by neutrophils may contribute to leukemia associated with inherited RUNX1 mutations.
    DOI:  https://doi.org/10.1182/bloodadvances.2019000785
  31. Nat Rev Gastroenterol Hepatol. 2020 Mar 19.
    Salas A, Hernandez-Rocha C, Duijvestein M, Faubion W, McGovern D, Vermeire S, Vetrano S, Vande Casteele N.
      Cytokines are involved in intestinal homeostasis and pathological processes associated with inflammatory bowel disease (IBD). The biological effects of cytokines, including several involved in the pathology of Crohn's disease and ulcerative colitis, occur as a result of receptor-mediated signalling through the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) DNA-binding families of proteins. Although therapies targeting cytokines have revolutionized IBD therapy, they have historically targeted individual cytokines, and an unmet medical need exists for patients who do not respond to or lose response to these treatments. Several small-molecule inhibitors of JAKs that have the potential to affect multiple pro-inflammatory cytokine-dependent pathways are in clinical development for the treatment of IBD, with one agent, tofacitinib, already approved for ulcerative colitis and several other agents with demonstrated efficacy in early phase trials. This Review describes the current understanding of JAK-STAT signalling in intestinal homeostasis and disease and the rationale for targeting this pathway as a treatment for IBD. The available evidence for the efficacy, safety and pharmacokinetics of JAK inhibitors in IBD as well as the potential approaches to optimize treatment with these agents, such as localized delivery or combination therapy, are also discussed.
    DOI:  https://doi.org/10.1038/s41575-020-0273-0
  32. J Leukoc Biol. 2020 Mar 23.
    Yang M, Tran L, Torrey H, Song Y, Perkins H, Case K, Zheng H, Takahashi H, Kuhtreiber WM, Faustman DL.
      Most approved cancer immunotherapies lack T-regulatory (Treg) or tumor specificity. TNF receptor 2 (TNFR2) antibody antagonism is emerging as an attractive immunotherapy due to its tumor microenvironment (TME) specificity. Here we show that the human TNFR2 receptor is overexpressed on both human tumor cells and on human tumor-residing Tregs, but negligibly expressed on beneficial T effectors (Teffs). Further, we found widespread, if variable, TNFR2 expression on 788 human tumor cell lines from diverse cancer tissues. These findings provided strong rationale for developing a targeted immunotherapy using a TNFR2 antibody antagonist. We designed a novel, human-directed TNFR2 antibody antagonist and tested it for function using three cell-based TME assays. The antagonist showed TME specificity by killing of TNFR2-expressing tumor cells and Tregs, but sparing Teffs, which proliferated. However, the antagonist shuffled between five isoforms, only one of which showed the desirable function. We designed and tested several new chimeric human versions of the antagonist, finding that the IgG2 isotype functioned better than the IgG1 isotype. To further improve function, we introduced targeted mutations to its amino acid sequence to stabilize the natural variability of the IgG2 isotype's hinge. Altogether, our findings suggest that optimal TNFR2 antagonists are of the human IgG2 isotype, have hinge stabilization, and have wide separation of antibody arms to bind to newly synthesized TNFR2 on rapidly growing tumor cells. Antagonistic antibodies with these characteristics, when bound to TNFR2, can form a nonsignaling cell surface dimer that functions with high TME specificity.
    Keywords:  3 cell-based assays; IgG2 isoform; hinge stabilization
    DOI:  https://doi.org/10.1002/JLB.5AB0320-415RRRRR
  33. Curr Biol. 2020 Mar 23. pii: S0960-9822(20)30031-2. [Epub ahead of print]30(6): R246-R248
    Cassetta L, Pollard JW.
      Cassetta and Pollard introduce tumor-associated macrophages and discuss their origin, diversity, function and plasticity.
    DOI:  https://doi.org/10.1016/j.cub.2020.01.031