Photoactivation of Innate Immunity Receptor TLR8 in Live Mammalian Cells by Genetic Encoding of Photocaged Tyrosine

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

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Stimulation of Innate Immunity byIn VivoCyclic di-GMP Synthesis Using Adenovirus

Clinical and Vaccine Immunology ◽

10.1128/cvi.00471-14 ◽

2014 ◽

Vol 21 (11) ◽

pp. 1550-1559 ◽

Cited By ~ 4

Author(s):

Benjamin J. Koestler ◽

Sergey S. Seregin ◽

David P. W. Rastall ◽

Yasser A. Aldhamen ◽

Sarah Godbehere ◽

...

Keyword(s):

Innate Immunity ◽

Mammalian Cells ◽

Immune Cell ◽

Innate Immune ◽

Host Cells ◽

Content Type ◽

Cytokines And Chemokines ◽

Novel Approach

ABSTRACTThe bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesizedin vivoby transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMPin vitroandin vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to aClostridium difficileantigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

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Genetic Encoding of Caged Cysteine and Caged Homocysteine in Bacterial and Mammalian Cells

ChemBioChem ◽

10.1002/cbic.201400073 ◽

2014 ◽

Vol 15 (12) ◽

pp. 1793-1799 ◽

Cited By ~ 33

Author(s):

Rajendra Uprety ◽

Ji Luo ◽

Jihe Liu ◽

Yuta Naro ◽

Subhas Samanta ◽

...

Keyword(s):

Mammalian Cells ◽

Genetic Encoding

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NF-κB Links CO2 Sensing to Innate Immunity and Inflammation in Mammalian Cells

The Journal of Immunology ◽

10.4049/jimmunol.1000701 ◽

2010 ◽

Vol 185 (7) ◽

pp. 4439-4445 ◽

Cited By ~ 64

Author(s):

Eoin P. Cummins ◽

Kathryn M. Oliver ◽

Colin R. Lenihan ◽

Susan F. Fitzpatrick ◽

Ulrike Bruning ◽

...

Keyword(s):

Innate Immunity ◽

Mammalian Cells

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Induction of Nitric Oxide Synthase in Anopheles stephensi by Plasmodium falciparum: Mechanism of Signaling and the Role of Parasite Glycosylphosphatidylinositols

Infection and Immunity ◽

10.1128/iai.73.5.2778-2789.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 2778-2789 ◽

Cited By ~ 88

Author(s):

Junghwa Lim ◽

D. Channe Gowda ◽

Gowdahalli Krishnegowda ◽

Shirley Luckhart

Keyword(s):

Nitric Oxide ◽

Innate Immunity ◽

Protein Kinase ◽

Nitric Oxide Synthase ◽

Insulin Signaling ◽

Mammalian Cells ◽

Anopheles Stephensi ◽

Midgut Epithelium ◽

Parasite Development ◽

Oxide Synthase

ABSTRACT Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h postinfection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that adversely impact parasite development. In mammals, P. falciparum glycosylphosphatidylinositols (PfGPIs) can induce NOS expression in immune and endothelial cells and are sufficient to reproduce the major effects of parasite infection. These effects are mediated in part by mimicry of insulin signaling by PfGPIs. In this study, we demonstrate that PfGPIs can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi Akt/protein kinase B and a pathway involving DSOR1, a mitogen-activated protein kinase kinase, and an extracellular signal-regulated kinase. However, despite the involvement of kinases that are also associated with insulin signaling in A. stephensi cells, signaling by P. falciparum and by PfGPIs is distinctively different from signaling by insulin. Therefore, although mimicry of insulin by PfGPIs appears to be restricted to mammalian hosts of P. falciparum, the conservation of PfGPIs as a prominent parasite-derived signal of innate immunity can now be extended to include Anopheles mosquitoes, indicating that parasite signaling of innate immunity is conserved in mosquito and mammalian cells.

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Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels–Alder Reactions

Journal of the American Chemical Society ◽

10.1021/ja302832g ◽

2012 ◽

Vol 134 (25) ◽

pp. 10317-10320 ◽

Cited By ~ 344

Author(s):

Kathrin Lang ◽

Lloyd Davis ◽

Stephen Wallace ◽

Mohan Mahesh ◽

Daniel J. Cox ◽

...

Keyword(s):

Mammalian Cells ◽

Specific Protein ◽

Diels Alder ◽

Protein Labeling ◽

Site Specific ◽

Genetic Encoding

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Control of Innate Immunity by Sialic Acids in the Nervous Tissue

International Journal of Molecular Sciences ◽

10.3390/ijms21155494 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5494 ◽

Cited By ~ 3

Author(s):

Huan Liao ◽

Christine Klaus ◽

Harald Neumann

Keyword(s):

Innate Immunity ◽

Sialic Acid ◽

Mammalian Cells ◽

Nervous Tissue ◽

Neurological Diseases ◽

Sialic Acids ◽

Factor H ◽

Complement Factor ◽

Sialidase Activity ◽

Microglial Phagocytosis

Sialic acids (Sias) are the most abundant terminal sugar residues of glycoproteins and glycolipids on the surface of mammalian cells. The nervous tissue is the organ with the highest expression level of Sias. The ‘sialylation’ of glycoconjugates is performed via sialyltransferases, whereas ‘desialylation’ is done by sialidases or is a possible consequence of oxidative damage. Sialic acid residues on the neural cell surfaces inhibit complement and microglial activation, as well as phagocytosis of the underlying structures, via binding to (i) complement factor H (CFH) or (ii) sialic acid-binding immunoglobulin-like lectin (SIGLEC) receptors. In contrast, activated microglial cells show sialidase activity that desialylates both microglia and neurons, and further stimulates innate immunity via microglia and complement activation. The desialylation conveys neurons to become susceptible to phagocytosis, as well as triggers a microglial phagocytosis-associated oxidative burst and inflammation. Dysfunctions of the ‘Sia–SIGLEC’ and/or ‘Sia–complement’ axes often lead to neurological diseases. Thus, Sias on glycoconjugates of the intact glycocalyx and its desialylation are major regulators of neuroinflammation.

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