scholarly journals Deep analysis of the USP18-dependent ISGylome and proteome unveils important roles for USP18 in tumour cell antigenicity and radiosensitivity

2020 ◽  
Author(s):  
Adan Pinto-Fernandez ◽  
Mariolina Salio ◽  
Tom Partridge ◽  
Jianzhou Chen ◽  
George Vere ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Cytotoxic T Lymphocytes ◽  
Negative Regulator ◽  
Type I ◽  
Conventional Chemotherapy ◽  
Pharmacological Target ◽  
Substrate Proteins ◽  
Signalling Cascade ◽  
Proteomic Techniques

AbstractThe deubiquitylating enzyme USP18 is a major negative regulator of the interferon (IFN) signalling cascade. IFN pathways contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy and are often deregulated in cancer. USP18 is the predominant human protease that cleaves interferon-stimulated gene ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. In this study, using advanced proteomic techniques, we have expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line (HAP1) treated with type I IFN. Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Our results suggest USP18 as a pharmacological target in cancer immunotherapy and radiotherapy.

scholarly journals Regulation of anaphylactic responses by phosphatidylinositol phosphate kinase type I α

2005 ◽  
Vol 201 (6) ◽  
pp. 859-870 ◽  
Author(s):  
Junko Sasaki ◽  
Takehiko Sasaki ◽  
Masakazu Yamazaki ◽  
Kunie Matsuoka ◽  
Choji Taya ◽  
...  
Keyword(s):  
Mast Cells ◽  
Actin Polymerization ◽  
Negative Regulator ◽  
Type I ◽  
Filamentous Actin ◽  
Critical Signal ◽  
Cell Functions ◽  
Phosphatidylinositol Phosphate

The membrane phospholipid phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] is a critical signal transducer in eukaryotic cells. However, the physiological roles of the type I phosphatidylinositol phosphate kinases (PIPKIs) that synthesize PI(4,5)P2 are largely unknown. Here, we show that the α isozyme of PIPKI (PIPKIα) negatively regulates mast cell functions and anaphylactic responses. In vitro, PIPKIα-deficient mast cells exhibited increased degranulation and cytokine production after Fcε receptor-I cross-linking. In vivo, PIPKIα−/− mice displayed enhanced passive cutaneous and systemic anaphylaxis. Filamentous actin was diminished in PIPKIα−/− mast cells, and enhanced degranulation observed in the absence of PIPKIα was also seen in wild-type mast cells treated with latrunculin, a pharmacological inhibitor of actin polymerization. Moreover, the association of FcεRI with lipid rafts and FcεRI-mediated activation of signaling proteins was augmented in PIPKIα−/− mast cells. Thus, PIPKIα is a negative regulator of FcεRI-mediated cellular responses and anaphylaxis, which functions by controlling the actin cytoskeleton and dynamics of FcεRI signaling. Our results indicate that the different PIPKI isoforms might be functionally specialized.

scholarly journals High doses of TGF-β potently suppress type I collagen via the transcription factor CUX1

2011 ◽  
Vol 22 (11) ◽  
pp. 1836-1844 ◽  
Author(s):  
Maria Fragiadaki ◽  
Tetsurou Ikeda ◽  
Abigail Witherden ◽  
Roger M Mason ◽  
David Abraham ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Transforming Growth Factor Β ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Type I ◽  
Aberrant Expression

Transforming growth factor-β (TGF-β) is an inducer of type I collagen, and uncontrolled collagen production leads to tissue scarring and organ failure. Here we hypothesize that uncovering a molecular mechanism that enables us to switch off type I collagen may prove beneficial in treating fibrosis. For the first time, to our knowledge, we provide evidence that CUX1 acts as a negative regulator of TGF-β and potent inhibitor of type I collagen transcription. We show that CUX1, a CCAAT displacement protein, is associated with reduced expression of type I collagen both in vivo and in vitro. We show that enhancing the expression of CUX1 results in effective suppression of type I collagen. We demonstrate that the mechanism by which CUX1 suppresses type I collagen is through interfering with gene transcription. In addition, using an in vivo murine model of aristolochic acid (AA)-induced interstitial fibrosis and human AA nephropathy, we observe that CUX1 expression was significantly reduced in fibrotic tissue when compared to control samples. Moreover, silencing of CUX1 in fibroblasts from kidneys of patients with renal fibrosis resulted in increased type I collagen expression. Furthermore, the abnormal CUX1 expression was restored by addition of TGF-β via the p38 mitogen-activated protein kinase pathway. Collectively, our study demonstrates that modifications of CUX1 expression lead to aberrant expression of type I collagen, which may provide a molecular basis for fibrogenesis.

scholarly journals NOD1 Participates in the Innate Immune Response Triggered by Hepatitis C Virus Polymerase

2016 ◽  
Vol 90 (13) ◽  
pp. 6022-6035 ◽  
Author(s):  
Serena Vegna ◽  
Damien Gregoire ◽  
Marie Moreau ◽  
Patrice Lassus ◽  
David Durantel ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Innate Immunity ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Inflammatory Response ◽  
Cellular Response ◽  
Pattern Recognition Receptors ◽  
Type I ◽  
Mitochondrial Antiviral Signaling

ABSTRACTHepatitis C virus (HCV) triggers innate immunity signaling in the infected cell. Replication of the viral genome is dispensable for this phenotype, and we along with others have recently shown that NS5B, the viral RNA-dependent RNA polymerase, synthesizes double-stranded RNA (dsRNA) from cellular templates, thus eliciting an inflammatory response, notably via activation of type I interferon and lymphotoxin β. Here, we investigated intracellular signal transduction pathways involved in this process. Using HepaRG cells, a model that largely recapitulates thein vivocomplexities of the innate immunity receptor signaling, we have confirmed that NS5B triggered increased expression of the canonical pattern recognition receptors (PRRs) specific for dsRNA, namely, RIG-I, MDA5, and Toll-like receptor 3 (TLR3). Unexpectedly, intracellular dsRNA also led to accumulation of NOD1, a receptor classically involved in recognition of bacterial peptidoglycans. NOD1 activation, confirmed by analysis of its downstream targets, was likely due to its interaction with dsRNA and was independent of RIG-I and mitochondrial antiviral signaling protein (MAVS/IPS-1/Cardif/VISA) signaling. It is likely to have a functional significance in the cellular response in the context of HCV infection since interference with the NOD1 pathway severely reduced the inflammatory response elicited by NS5B.IMPORTANCEIn this study, we show that NOD1, a PRR that normally senses bacterial peptidoglycans, is activated by HCV viral polymerase, probably through an interaction with dsRNA, suggesting that NOD1 acts as an RNA ligand recognition receptor. In consequence, interference with NOD1-mediated signaling significantly weakens the inflammatory response to dsRNA. These results add a new level of complexity to the understanding of the cross talk between different classes of pattern recognition receptors and may be related to certain complications of chronic hepatitis C virus infection.

scholarly journals HIVEP1 Is a Negative Regulator of NF-κB That Inhibits Systemic Inflammation in Sepsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Hisatake Matsumoto ◽  
Brendon P. Scicluna ◽  
Kin Ki Jim ◽  
Fahimeh Falahi ◽  
Wanhai Qin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Negative Regulator ◽  
Type I ◽  
Promoter Regions ◽  
Monocytic Cells ◽  
Site Analysis ◽  
Enhancer Binding Protein ◽  
Transcriptomic Changes

Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.

scholarly journals LncRNAMalat1inhibition of TDP43 cleavage suppresses IRF3-initiated antiviral innate immunity

2020 ◽  
Vol 117 (38) ◽  
pp. 23695-23706 ◽  
Author(s):  
Wei Liu ◽  
Ziqiao Wang ◽  
Lun Liu ◽  
Zongheng Yang ◽  
Shuo Liu ◽  
...  
Keyword(s):  
Viral Infection ◽  
Lupus Erythematosus ◽  
Mononuclear Cells ◽  
Negative Regulator ◽  
Type I ◽  
Type I Ifn ◽  
Peripheral Blood Mononuclear ◽  
Interactive Network ◽  
Antiviral Innate Immunity

Long noncoding RNAs (lncRNAs) involved in the regulation of antiviral innate immune responses need to be further identified. By functionally screening the lncRNAs in macrophages, here we identified lncRNAMalat1, abundant in the nucleus but significantly down-regulated after viral infection, as a negative regulator of antiviral type I IFN (IFN-I) production.Malat1directly bound to the transactive response DNA-binding protein (TDP43) in the nucleus and prevented activation of TDP43 by blocking the activated caspase-3-mediated TDP43 cleavage to TDP35. The cleaved TDP35 increased the nuclear IRF3 protein level by binding and degradingRbck1pre-mRNA to prevent IRF3 proteasomal degradation upon viral infection, thus selectively promoting antiviral IFN-I production. Deficiency ofMalat1enhanced antiviral innate responses in vivo, accompanying the increased IFN-I production and reduced viral burden. Importantly, the reducedMALAT1, augmented IRF3, and increasedIFNAmRNA were found in peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients. Therefore, the down-regulation ofMALAT1in virus-infected cells or in human cells from autoimmune diseases will increase host resistance against viral infection or lead to autoinflammatory interferonopathies via the increased type I IFN production. Our results demonstrate that the nuclearMalat1suppresses antiviral innate responses by targeting TDP43 activation via RNA-RBP interactive network, adding insight to the molecular regulation of innate responses and autoimmune pathogenesis.

scholarly journals Saccharomyces cerevisiaeRot1 Is an Essential Molecular Chaperone in the Endoplasmic Reticulum

2008 ◽  
Vol 19 (8) ◽  
pp. 3514-3525 ◽  
Author(s):  
Masato Takeuchi ◽  
Yukio Kimata ◽  
Kenji Kohno
Keyword(s):  
Molecular Chaperones ◽  
Secretory Pathway ◽  
Temperature Sensitive ◽  
Type I ◽  
Accelerated Degradation ◽  
Transient Interaction ◽  
Protein Functions ◽  
Substrate Proteins

Molecular chaperones prevent aggregation of denatured proteins in vitro and are thought to support folding of diverse proteins in vivo. Chaperones may have some selectivity for their substrate proteins, but knowledge of particular in vivo substrates is still poor. We here show that yeast Rot1, an essential, type-I ER membrane protein functions as a chaperone. Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation. In vivo, the rot1-2 mutation caused accelerated degradation of five proteins in the secretory pathway via ER-associated degradation, resulting in a decrease in their cellular levels. Furthermore, we demonstrate a physical and probably transient interaction of Rot1 with four of these proteins. Collectively, these results indicate that Rot1 functions as a chaperone in vivo supporting the folding of those proteins. Their folding also requires BiP, and one of these proteins was simultaneously associated with both Rot1 and BiP, suggesting that they can cooperate to facilitate protein folding. The Rot1-dependent proteins include a soluble, type I and II, and polytopic membrane proteins, and they do not share structural similarities. In addition, their dependency on Rot1 appeared different. We therefore propose that Rot1 is a general chaperone with some substrate specificity.

scholarly journals CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1818-1828 ◽  
Author(s):  
Cyndi Wong ◽  
Yong Liu ◽  
Jana Yip ◽  
Rochna Chand ◽  
Janet L. Wee ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Tyrosine Phosphatase ◽  
Negative Regulator ◽  
Surface Glycoprotein ◽  
Type I ◽  
Wild Type ◽  
Glycoprotein Vi ◽  
Selective Ligand

Abstract Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)–γ-chain. ceacam1−/− platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRP-mediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1−/− mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1−/− mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1−/− mice showed a reversal in the more stable thrombus growth phenotype. ceacam1−/− mice were more susceptible to type I collagen–induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo.

scholarly journals The RNA helicase DDX5 promotes viral infection via regulating N6-methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity

2021 ◽  
Vol 17 (4) ◽  
pp. e1009530
Author(s):  
Jian Xu ◽  
Yunhong Cai ◽  
ZhenBang Ma ◽  
Bo Jiang ◽  
Wenxiao Liu ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Viral Infection ◽  
Nuclear Export ◽  
Rna Virus ◽  
Negative Regulator ◽  
Innate Response ◽  
Viral Propagation ◽  
Dead Box ◽  
Antiviral Innate Immunity

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3–METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.

scholarly journals T Cell-Intrinsic CDK6 Is Dispensable for Anti-Viral and Anti-Tumor Responses In Vivo

2021 ◽  
Vol 12 ◽  
Author(s):  
Klara Klein ◽  
Agnieszka Witalisz-Siepracka ◽  
Dagmar Gotthardt ◽  
Benedikt Agerer ◽  
Felix Locker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Negative Regulator ◽  
Fine Tuning ◽  
Type I ◽  
Cell Functions ◽  
Intrinsic Loss

The cyclin-dependent kinase 6 (CDK6) regulates the transition through the G1-phase of the cell cycle, but also acts as a transcriptional regulator. As such CDK6 regulates cell survival or cytokine secretion together with STATs, AP-1 or NF-κB. In the hematopoietic system, CDK6 regulates T cell development and promotes leukemia and lymphoma. CDK4/6 kinase inhibitors are FDA approved for treatment of breast cancer patients and have been reported to enhance T cell-mediated anti-tumor immunity. The involvement of CDK6 in T cell functions remains enigmatic. We here investigated the role of CDK6 in CD8+ T cells, using previously generated CDK6 knockout (Cdk6-/-) and kinase-dead mutant CDK6 (Cdk6K43M) knock-in mice. RNA-seq analysis indicated a role of CDK6 in T cell metabolism and interferon (IFN) signaling. To investigate whether these CDK6 functions are T cell-intrinsic, we generated a T cell-specific CDK6 knockout mouse model (Cdk6fl/fl CD4-Cre). T cell-intrinsic loss of CDK6 enhanced mitochondrial respiration in CD8+ T cells, but did not impact on cytotoxicity and production of the effector cytokines IFN-γ and TNF-α by CD8+ T cells in vitro. Loss of CDK6 in peripheral T cells did not affect tumor surveillance of MC38 tumors in vivo. Similarly, while we observed an impaired induction of early responses to type I IFN in CDK6-deficient CD8+ T cells, we failed to observe any differences in the response to LCMV infection upon T cell-intrinsic loss of CDK6 in vivo. This apparent contradiction might at least partially be explained by the reduced expression of Socs1, a negative regulator of IFN signaling, in CDK6-deficient CD8+ T cells. Therefore, our data are in line with a dual role of CDK6 in IFN signaling; while CDK6 promotes early IFN responses, it is also involved in the induction of a negative feedback loop. These data assign CDK6 a role in the fine-tuning of cytokine responses.

scholarly journals The Superimposed Deubiquitination Effect of OTULIN and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Nsp11 Promotes Multiplication of PRRSV

2018 ◽  
Vol 92 (9) ◽  
Author(s):  
Yanxin Su ◽  
Peidian Shi ◽  
Lilin Zhang ◽  
Dong Lu ◽  
Chengxue Zhao ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Cell Model ◽  
Nuclear Factor Κb ◽  
Cellular Protein ◽  
Type I Interferons ◽  
Respiratory Syndrome Virus ◽  
Type I ◽  
Cooperative Effects ◽  
Superimposed Effect

ABSTRACTLinear ubiquitination plays an important role in the regulation of the immune response by regulating nuclear factor κB (NF-κB). The linear ubiquitination-specific deubiquitinase ovarian tumor domain deubiquitinase with linear linkage specificity (OTULIN) can control the immune signaling transduction pathway by restricting the Met1-linked ubiquitination process. In our study, the porcine OTLLIN gene was cloned and deubiquitin functions were detected in a porcine reproductive and respiratory syndrome virus (PRRSV)-infected-cell model. PRRSV infection promotes the expression of the OTULIN gene; in turn, overexpression of OTULIN contributes to PRRSV proliferation. There is negative regulation of innate immunity with OTULIN during viral infection. The cooperative effects of swine OTULIN and PRRSV Nsp11 potentiate the ability to reduce levels of cellular protein ubiquitin associated with innate immunity. Importantly, PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to enhance its ability to remove linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I interferons (IFNs). Our report presents a new model of virus utilization of the ubiquitin-protease systemin vivofrom the perspective of the viral proteins that interact with cell deubiquitination enzymes, providing new ideas for prevention and control of PRRSV.IMPORTANCEDeubiquitination effects of swine OTULIN were identified. The interaction between porcine OTULIN and PRRSV Nsp11 is dependent on the OTU domain. PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to promote removal of linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I IFNs.

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