scholarly journals Palmitoylation of the Bovine Foamy Virus Envelope Glycoprotein Is Required for Viral Replication

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 31
Author(s):  
Keli Chai ◽  
Zhaohuan Wang ◽  
Yali Xu ◽  
Junshi Zhang ◽  
Juan Tan ◽  
...  
Keyword(s):  
Viral Replication ◽  
Foamy Virus ◽  
Critical Role ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Minor Effect ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Virus Envelope ◽  
Bovine Foamy Virus

Membrane proteins of enveloped viruses have been reported to undergo palmitoylation, a post-translational modification often having a critical role in the function of these viral proteins and hence viral replication. In this study, we report that the foamy virus (FV) envelope (Env) glycoprotein is palmitoylated. Specifically, we found that bovine foamy virus (BFV) Env (BEnv) is palmitoylated at amino acid positions C58 and C59 by BDHHC3 and BDHHC20 in a DHHC motif-dependent manner. In addition, mutations C58S and C58/59S significantly decrease cell surface expression of BEnv, subviral particle (SVP) egress, and its membrane fusion activity, thus ultimately inhibiting BFV replication. The C59S mutation exerts a minor effect in this regard. Taken together, these data demonstrate that the function of BEnv in the context of BFV replication is under the regulation of palmitoylation.

scholarly journals Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region

2021 ◽  
Vol 22 (19) ◽  
pp. 10207
Author(s):  
Julien Vitry ◽  
Guillaume Paré ◽  
Andréa Murru ◽  
Xavier Charest-Morin ◽  
Halim Maaroufi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Activation ◽  
Secretory Pathway ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Inhibitory Receptor ◽  
Lectin Receptors ◽  
Stalk Domain

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

scholarly journals Elongation of the Cytoplasmic Tail Interferes with the Fusion Activity of Influenza Virus Hemagglutinin

1998 ◽  
Vol 72 (5) ◽  
pp. 3554-3559 ◽  
Author(s):  
Masanobu Ohuchi ◽  
Christian Fischer ◽  
Reiko Ohuchi ◽  
Astrid Herwig ◽  
Hans-Dieter Klenk
Keyword(s):  
Intracellular Transport ◽  
Critical Role ◽  
Simian Virus 40 ◽  
Cytoplasmic Tail ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Simian Virus ◽  
Cell Surface Expression ◽  
Erythrocyte Membranes ◽  
Fusion Pores

ABSTRACT The hemagglutinin (HA) of fowl plague virus was lengthened and shortened by site-specific mutagenesis at the cytoplasmic tail, and the effects of these modifications on HA functions were analyzed after expression from a simian virus 40 vector. Elongation of the tail by the addition of one to six histidine (His) residues did not interfere with intracellular transport, glycosylation, proteolytic cleavage, acylation, cell surface expression, and hemadsorption. However, the ability to induce syncytia at a low pH decreased dramatically depending on the number of His residues added. Partial fusion (hemifusion), assayed by fluorescence transfer from octadecylrhodamine-labeled erythrocyte membranes, was also reduced, but even with the mutant carrying six His residues, significant transfer was observed. However, when the formation of fusion pores was examined with hydrophilic fluorescent calcein, transfer from erythrocytes to HA-expressing cells was not observed with the mutant carrying six histidine residues. The addition of different amino acids to the cytoplasmic tail of HA caused an inhibitory effect similar to that caused by the addition of His. On the other hand, a mutant lacking the cytoplasmic tail was still able to fuse at a reduced level. These results demonstrate that elongation of the cytoplasmic tail interferes with the formation and enlargement of fusion pores. Thus, the length of the cytoplasmic tail plays a critical role in the fusion process.

scholarly journals Two N-Linked Glycans Differentially Control Maturation, Trafficking and Proteolysis, but not Activity of the IL-11 Receptor

2018 ◽  
Vol 45 (5) ◽  
pp. 2071-2085 ◽  
Author(s):  
Maria Agthe ◽  
Yvonne Garbers ◽  
Joachim Grötzinger ◽  
Christoph Garbers
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Proteolytic Processing ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Post Translational Modification ◽  
Glycosylation Sites ◽  
D2 Domain

Background/Aims: The cytokine interleukin-11 (IL-11) has important pro- and anti-inflammatory functions. It activates its target cells through binding to the IL-11 receptor (IL-11R), and the IL-11/IL-11R complex recruits a homodimer of glycoprotein 130 (gp130). N-linked glycosylation, a post-translational modification where complex oligosaccharides are attached to the side chain of asparagine residues, is often important for stability, folding and biological function of cytokine receptors. Methods: We generated different IL-11R mutants via site-directed mutagenesis and analyzed them in different cell lines via Western blot, flow cytometry, confocal microscopy and proliferation assays. Results: In this study, we identified two functional N-glycosylation sites in the D2 domain of the IL-11R at N127 and N194. While mutation of N127Q only slightly affects cell surface expression of the IL-11R, mutation of N194Q broadly prevents IL-11R appearance at the plasma membrane. Accordingly, IL-11R mutants lacking N194 are retained within the ER, whereas the N127 mutant is transported through the Golgi complex to the cell surface, uncovering a differential role of the two N-glycan sequons for IL-11R maturation. Interestingly, IL-11R mutants devoid of one or both N-glycans are still biologically active. Furthermore, the IL-11RN127Q/N194Q mutant shows no inducible shedding by ADAM10, but is rather constitutively released into the supernatant. Conclusion: Our results show that the two N-glycosylation sites differentially influence stability and proteolytic processing of the IL-11R, but that N-linked glycosylation is not a prerequisite for IL-11 signaling.

scholarly journals All-Trans Retinoic Acid Enhances Bacterial Flagellin-Stimulated Proinflammatory Responses in Human Monocyte THP-1 Cells by Upregulating CD14

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Thi Xoan Hoang ◽  
Jong Hyeok Jung ◽  
Jae Young Kim
Keyword(s):  
Retinoic Acid ◽  
Cell Surface ◽  
Human Monocyte ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Atra Treatment ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Proinflammatory Responses

All-trans retinoic acid (ATRA), an active form of vitamin A, exerts immunomodulatory functions. In this study, we examined the immune potentiating effect of ATRA on bacterial flagellin-induced NF-κB activation and proinflammatory cytokine production in human monocytic cell line THP-1. ATRA treatment significantly enhanced the flagellin-induced NF-κB/AP-1 activity in THP-1 via the RAR/RXR pathway. Similarly, ATRA enhanced the expression and production of TNF-α and IL-1β in THP-1 cells upon flagellin challenge. The cell surface expression of toll-like receptor 5 (TLR5), which is the receptor for bacterial flagellin, was significantly reduced by ATRA in a concentration- and time-dependent manner. To determine the mechanisms underlying the ATRA-enhanced immune response against bacterial flagellin despite the reduced cell surface expression of TLR5 in ATRA-treated THP-1, we examined the cell surface expression of CD14, which has been proposed to be a TLR co-receptor that enhances the response to microbial components. The cell surface expression of CD14 was significantly enhanced by ATRA treatment, especially in the presence of flagellin. Anti-CD14 antibody treatment prior to ATRA and flagellin treatments completely abolished ATRA-enhanced TNF-α and IL-1β production. Our results suggest that ATRA enhances flagellin-stimulated proinflammatory responses in human monocyte THP-1 cells by upregulating CD14 in a RAR/RXR-dependent manner.

scholarly journals DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

2020 ◽  
Author(s):  
Kelly E. Leon ◽  
Raquel Buj ◽  
Elizabeth Lesko ◽  
Erika S. Dahl ◽  
Chi-Wei Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Epigenetic Regulation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Inhibition Of Proliferation ◽  
Cycle Arrest ◽  
Secretory Phenotype ◽  
Negative Side Effects

AbstractCellular senescence is characterized as a stable cell cycle arrest that can occur as a stress response associated with oncogenic activation, termed oncogene-induced senescence (OIS). Cells undergoing OIS acquire a unique microenvironment termed the senescence-associated secretory phenotype (SASP), which can be both beneficial and detrimental in a context-dependent manner. Additionally, senescent cells are characterized by robust changes in their epigenome. Here, we globally assessed the histone landscape of cells induced to senesce by oncogenic RAS and discovered a novel epigenetic regulatory mechanism of the key SASP regulator IL1A. OIS cells displayed increased di- and tri-methylation of histone H3 lysine 79 (H3K79me2/3), two active histone marks. Depletion of the H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) during OIS resulted in decreased H3K79me2/3 occupancy at the IL1A gene locus, which corresponded to decreased IL1A mRNA and cell surface expression. Decreased expression and secretion of downstream cytokines without a change in senescence markers were also observed upon DOT1L depletion. Overexpression of DOT1L increased H3K79me2/3 occupancy at the IL1A locus and upregulated the SASP, indicating that DOT1L is both necessary and sufficient for SASP gene expression. Mechanistically, we found that STING, an essential mediator of SASP transcription, is upstream of DOT1L in the epigenetic regulation of the SASP. Together, our studies establish DOT1L as an epigenetic regulator of the SASP whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.

scholarly journals Distinct Cell Surface Expression Patterns of N-Glycosylation Site Mutants of AMPA-Type Glutamate Receptor under the Homo-Oligomeric Expression Conditions

2020 ◽  
Vol 21 (14) ◽  
pp. 5101
Author(s):  
Jyoji Morise ◽  
Saki Yamamoto ◽  
Ryosuke Midorikawa ◽  
Kogo Takamiya ◽  
Motohiro Nonaka ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glutamate Receptor ◽  
Expression Patterns ◽  
Surface Expression ◽  
Glycosylation Site ◽  
Synaptic Activity ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Distinct Cell

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1–4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits’ ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.

Abstract 5309: beta-Arrestin 1 (bArr1) and Ankyrin 2 (ANK2) Regulate Cav1.2 Trafficking through Complex G Protein Mediated Signaling

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Maria A Diverse-Pierluissi ◽  
Rachele Lipsky ◽  
Akil A Puckerin ◽  
Joanne Stocks ◽  
Vivek Patel ◽  
...  
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
Surface Expression ◽  
Signaling Molecules ◽  
Cell Surface Expression ◽  
Receptor Subtype ◽  
Membrane Insertion ◽  
Dependent Manner ◽  
Kinase Inhibition ◽  
Rat Cardiomyocytes

Mechanisms governing the trafficking of cardiac voltage dependent calcium channels (Ca v 1.2) remain largely unknown. We have previously shown that neuronal calcium channels are internalized into clathrin coated vesicles upon G protein coupled receptor (GPCR) activation. Here, we hypothesize that GPCR mediated signaling regulate Ca v 1.2 trafficking and surface expression through interaction with cytoskeletal and signaling molecules. Methods : Sequential immunoprecipitation in adult rat cardiomyocytes was used to investigate the interaction between Ca v 1.2, cytoskeletal and signaling molecules. Live cell imaging and immunofluorescence microscopy were used to measure changes in the cellular localization of Ca v 1.2 and these proteins upon activation of GPCR signaling. Results : Cav1.2 interacts with ANK2, βArr1 and spectrin. Sustained β adrenergic receptor (βAR) activation increases Cav1.2/spectrin and decreases Ca v 1.2/ANK2 and Ca v 1.2/βArr1 interactions. Sustained βAR activation induces rapid (<5 min) Cav1.2 internalization and dissociation from ANK2 as Ca v 1.2/ANK2 co-localization is markedly reduced (−58% p<0.01) in isoproterenol treated myocytes. A cell permeant peptide (1.4 μg/ml) that disrupts Ca v 1.2/ANK2 interaction decreases (−40±12%, p<0.01) Cav1.2 cell surface expression. Pretreatment with pertussis toxin prevents Ca v 1.2 internalization suggesting that G i/o mediates this response. Similarly, Src kinase inhibition reduces (by 90%) Ca v 1.2 internalization. Differential labeling of pre-existing Ca v 1.2 at the cell membrane and newly inserted Ca v 1.2 using fluorophore conjugated dihydropyridines reveal that activation of G i/o proteins underlies Ca v 1.2 internalization and insertion. In contrast, activation of the muscarinic M2 receptor (10μM carbachol) causes only insertion of new channels (n=14) and fails to alter the association of Ca v 1.2 with ANK2 and spectrin. Finally PI3 kinase inhibition (10nM wortmannin) prevents Ca v 1.2 membrane insertion. Conclusions : Our findings reveal novel signaling mechanisms that regulate Ca v 1.2 trafficking, internalization and membrane insertion in a receptor subtype dependent manner. These mechanisms may be central to pathologies associated with a hyperadrenergic state. This research has received full or partial funding support from the American Heart Association, AHA National Center.

scholarly journals Bovine CD18 Is Necessary and Sufficient To Mediate Mannheimia (Pasteurella) haemolytica Leukotoxin-Induced Cytolysis

2002 ◽  
Vol 70 (9) ◽  
pp. 5058-5064 ◽  
Author(s):  
M. S. Deshpande ◽  
T. C. Ambagala ◽  
A. P. N. Ambagala ◽  
M. E. Kehrli ◽  
S. Srikumaran
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Surface Proteins ◽  
Polymorphonuclear Neutrophils ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Dependent Manner ◽  
Β Subunit ◽  
Concentration Dependent Manner ◽  
Necessary And Sufficient

ABSTRACT Leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica is an RTX toxin which is specific for ruminant leukocytes. Lkt binds to β2 integrins on the surface of bovine leukocytes. β2 integrins have a common β subunit, CD18, that associates with three distinct α chains, CD11a, CD11b, and CD11c, to give rise to three different β2 integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (CR4), respectively. Our earlier studies revealed that Lkt binds to all three β2 integrins, suggesting that the common β subunit, CD18, may be the receptor for Lkt. In order to unequivocally elucidate the role of bovine CD18 as a receptor for Lkt, a murine cell line nonsusceptible to Lkt (P815) was transfected with cDNA for bovine CD18. One of the transfectants, 2B2, stably expressed bovine CD18 on the cell surface. The 2B2 transfectant was effectively lysed by Lkt in a concentration-dependent manner, whereas the P815 parent cells were not. Immunoprecipitation of cell surface proteins of 2B2 with monoclonal antibodies specific for bovine CD18 or murine CD11a suggested that bovine CD18 was expressed on the cell surface of 2B2 as a heterodimer with murine CD11a. Expression of bovine CD18 and the Lkt-induced cytotoxicity of 2B2 cells were compared with those of bovine polymorphonuclear neutrophils and lymphocytes. There was a strong correlation between cell surface expression of bovine CD18 and percent cytotoxicity induced by Lkt. These results indicate that bovine CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells.

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