The 24 kDa <i>N</i>‐terminal sub‐domain of the DNA gyrase B protein binds coumarin drugs

Gilbert, E J; Maxwell, Anthony

doi:10.1111/j.1365-2958.1994.tb01026.x

Cited by 76 publications

(73 citation statements)

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“…2B). Addition of a gyrase dimerization-inducing agent, coumermycin A1 (35), to the GYRB-TM-TLR4-expressing cells did not significantly increase the cellular activation (Fig. 2C), indicating that dimers or higher-order oligomers of the chimeras must have already been formed even in the absence of coumermycin A1.…”

Section: The Tlr4 Ectodomain Has Additional Roles Besides Providing Amentioning

confidence: 99%

“…This is supported by the previous report on the addition of the CD4 dimerization domain to the transmembrane and cytoplasmic domains of TLR4, which also resulted in constitutive activity of CD4-TLR4 chimeras (17). To rule out the effect of intrinsic dimerization of ectodomain replacements, MD-2 and CD14 were substituted by the monomeric protein mCherry (34) or the 24-kDa N-terminal fragment of Escherichia coli gyrase B (GYRB24), which has previously been used as a fusion partner in coumermycininduced dimerization (35,36). The CHERRY-TM-TLR4 and GYRB24-TM-TLR4 constructs are schematically represented in Fig.…”

Section: The Tlr4 Ectodomain Has Additional Roles Besides Providing Amentioning

confidence: 99%

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The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

Panter

Jerala

2011

Journal of Biological Chemistry

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Toll-like receptor 4 (TLR4) is involved in activation of the innate immune response in a large number of different diseases.Despite numerous studies, the role of separate domains of TLR4 in the regulation of receptor activation is poorly understood. Replacement of the TLR4 ectodomain with LPS-binding proteins MD-2 or CD14 resulted in a robust ligand-independent constitutive activation comparable with the maximal stimulation of the receptor with LPS. The same effect was achieved by the replacement of the ectodomain with a monomeric fluorescent protein or a 24-kDa gyrase B fragment. This demonstrates an intrinsic dimerization propensity of the transmembrane and cytoplasmic domains of TLR4 and reveals a previously unknown function of the ectodomain in inhibiting spontaneous receptor dimerization. Constitutive activation was abolished by the replacement of the ectodomain by a bulkier protein ovalbumin. N-terminal deletion variants of TLR4 revealed that the smallest segment of the ectodomain that already prevents constitutive activity comprises only 90 residues (542 to 631) of the total 608 residues. We conclude that TLR4 represents a receptor with a low threshold of activation that can be rapidly activated by the release of inhibition exerted by its ectodomain. This is important for the sensitivity of TLR4 to activation by different agonists. The TLR4 ectodomain has multiple roles in enabling ligand regulated activation, providing proper localization while serving as an inhibitor to prevent spontaneous, ligand-independent dimerization.Pattern recognition receptors are proteins expressed by cells of the innate immune system and act as a first line of host defense against invading microorganisms, recognizing diverse but highly conserved structural pathogen-associated molecular patterns of bacteria, fungi, and viruses (1). Activation of pattern recognition receptors by their respective ligands triggers an immediate immune response, characterized by proinflammatory cytokine production (1, 2). Toll-like receptors (TLRs) 2 are a family of pattern recognition receptors central to the vertebrate innate immune response. Activation of TLRs links innate and adaptive immunity through proinflammatory signaling and induction of costimulatory molecules on immune cells (3, 4). However, activation of TLRs has to be a tightly regulated process. TLRs have to be able to mount an immediate immune response upon binding of the agonist, whereas in the absence of the ligand it is imperative that they remain in an inactive state to prevent unwanted activation that may lead to excessive inflammation and autoimmune disease (5). This is especially important for TLR4, the cellular receptor for LPS, a molecular signature of the outer cell membrane of Gram-negative bacteria (6). Activation of TLR4 by LPS affects the regulation of more than 1000 genes (7). Additionally, TLR4 signaling has been implicated in sterile inflammation through activation of the receptor by endogenous ligands (8 -14).TLR4 is a type I transmembrane glycoprotein. The ectodoma...

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Section: The Tlr4 Ectodomain Has Additional Roles Besides Providing Amentioning

confidence: 99%

Section: The Tlr4 Ectodomain Has Additional Roles Besides Providing Amentioning

confidence: 99%

The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

Panter

Jerala

2011

Journal of Biological Chemistry

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“…xanthus GyrB) thought to form a flexible link between the two N-terminal subdomains (Gilbert & Maxwell, 1994 ; Fig. 2).…”

Section: Discussionmentioning

confidence: 99%

Molecular analysis of the DNA gyrB gene from Myxococcus xanthus

et al. 1998

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DNA gyrase, an essential type II topoisomerase, mediates negative supercoiling of the bacterial chromosome, thereby affecting the processes of DNA replication, transcription, recombination and repair. The gyrB gene from the Gram-negative soil bacterium Myxococcus xanthus was sequenced. The sequence predicts a protein of 81 5 amino acid residues displaying significant homology to all known GyrB proteins. A 6-His-GyrB fusion protein was overexpressed in Escherichia coli and purified to near homogeneity using affinity chromatography on Ni-nitrilotriacetic acid-agarose and novobiocin-Sepharose columns. The fusion protein bound novobiocin and cross-reacted with anti-€. coli GyrB antibodies, indicating structural and functional similarities to the E. coli DNA GyrB. The gene was mapped to the region of the origin of replication (oriC) of M. xanthus.

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“…This means of producing "on-demand" gene induction adds another layer of complexity in that it is controlled through two antibiotics, one of which is a potent inducer and another which has antagonistic function [85]. Coumermycin acts through binding of the B subunit of bacterial DNA gyrase, causing dimerization and thereby inhibiting growth [35]. The expression system exploits this function of the antibiotic by fusing the B subunit of bacterial DNA gyrase to the λ repressor DNA-binding domain.…”

Section: Coumermycin/novobiocin-regulated Gene Expressionmentioning

confidence: 99%

Selected technologies to control genes and their products for experimental and clinical purposes

Alexander

Booy

Xiao

et al. 2007

Arch. Immunol. Ther. Exp.

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Abstract"On-demand" regulation of gene expression is a powerful tool to elucidate the functions of proteins and biologically-active RNAs. We describe here three different approaches to the regulation of expression or activity of genes or proteins. Promoter-based regulation of gene expression was among the most rapidly developing techniques in the 1980s and 1990s. Here we provide basic information and also some characteristics of the metallothionein-promoter-based system, the tet-off system, Muristerone-A-regulated expression through the ecdysone response element, RheoSwitch ® , coumermycin/novobiocin-regulated gene expression, chemical dimerizer-based promoter activation systems, the "Dual Drug Control" system, "constitutive androstane receptor"-based regulation of gene expression, and RU486/mifepristone-driven regulation of promoter activity. A large part of the review concentrates on the principles and usage of various RNA interference techniques (RNAi: siRNA, shRNA, and miRNA-based methods). Finally, the last part of the review deals with historically the oldest, but still widely used, methods of temperature-dependent regulation of enzymatic activity or protein stability (temperature--sensitive mutants). Due to space limitations we do not describe in detail but just mention the tet-regulated systems and also fusion-protein-based regulation of protein activity, such as estrogen-receptor fusion proteins. The information provided below is aimed to assist researchers in choosing the most appropriate method for the planned development of experimental systems with regulated expression or activity of studied proteins.Key words: Dicer, Drosha, FKBP, gene silencing, Mifepristone, mutagensis, N-degron, NF-κB, RISC. *Both authors contributed equally to this review manuscript.Abbreviations: CAR -constitutive androstane receptor, dsRNA -double-stranded RNA, DHFR -dihydrofolate reductase, EcR -ecdysone receptor, FKBP -FK506-binding protein, FRAP -FKBP-rapamycin-associated protein, Hsp-90 -heat shock protein 90, MRE -metal-responsive elements, miRNAs -microRNAs, MCM -minichromosome maintenance, NF-κB -nuclear factor κB, PBREM -phenobarbital-responsive enhancer module, tetR -tetracycline-dependent repressor, tTAtetracycline-controlled transactivator, tetO -tetracycline operator, TK -thymidine kinase, RXR -retinoid X receptor, RNAi -RNA interference, RISC -RNA-induced silencing complex, shRNAs -short hairpin RNAs, siRNAs -small interfering RNAs, td -temperature-inducible degron, ts -temperature-sensitive, VP16 -virion protein 16 of herpes simplex virus.

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The 24 kDa N‐terminal sub‐domain of the DNA gyrase B protein binds coumarin drugs

Cited by 76 publications

References 39 publications

The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

Molecular analysis of the DNA gyrB gene from Myxococcus xanthus

Selected technologies to control genes and their products for experimental and clinical purposes

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