Role of cysteine62in DNA recognition by the P50 subunit of NF-xB

Matthews, James R.; Kaszubska, Wiweka; Turcatti, Gerardo; Wells, Timothy N.C.; Hay, Ronald T.

doi:10.1093/nar/21.8.1727

Cited by 146 publications

(97 citation statements)

References 32 publications

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“…Acrolein and crotonaldehyde reacted with cysteine, arginine, and histidine residues on the p50 subunit of NF-B and inhibited p50 DNA binding, whereas the saturated aldehydes did not react with these residues and had no effect on DNAbinding activity. Cys-61 is known to play a critical role in p50 DNA binding activity (42,43). Replacement of this cysteine residue with a serine increases the dissociation rate with DNA by Ͼ500-fold (42).…”

Section: Discussionmentioning

confidence: 99%

“…Cys-61 is known to play a critical role in p50 DNA binding activity (42,43). Replacement of this cysteine residue with a serine increases the dissociation rate with DNA by Ͼ500-fold (42). Both acrolein and crotonaldehyde alkylated Cys-61, but a comparison of precursor ion peak areas in the ion trap MS scan suggested that acrolein reacted with twice as much Cys-61 on a molar ratio.…”

Section: Discussionmentioning

confidence: 99%

“…Fig. 6A shows the amino acid sequence of the peptide 59 -76, which contains a cysteine residue at position 61 that is known to mediate DNA binding (42). The observed mass of the singly charged [MϩH] ϩ peptide of 59 -76 increased from the predicted mass-to-charge ratio (m/z) of 1774.83 (Fig.…”

Section: Inhibition Of Recombinant P50 Dna Binding By Cigarette Smokementioning

confidence: 98%

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Acrolein Inhibits Cytokine Gene Expression by Alkylating Cysteine and Arginine Residues in the NF-κB1 DNA Binding Domain

Lambert¹,

Li²,

Jonscher

et al. 2007

Journal of Biological Chemistry

107

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Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The ␣,␤-unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of interleukin-2 (IL-2), IL-10, granulocyte-macrophage colony-stimulating factor, interferon-␥, and tumor necrosis factor-␣ by human T cells but did not inhibit production of IL-8. The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) in cigarette smoke were inactive. Acrolein inhibited induction of NF-B DNA binding activity after mitogenic stimulation of T cells but had no effect on induction of NFAT or AP-1. Acrolein inhibited NF-B1 (p50) binding to the IL-2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2000-fold more potent than crotonaldehyde in blocking DNA binding to an NF-B consensus sequence. Matrix-assisted laser desorption/ionization time-offlight and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys-61 and Arg-307) in the DNA binding domain. Crotonaldehyde reacted with Cys-61, but not Arg-307, whereas the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor.Cigarette smoke produces profound suppression of pulmonary immunity, resulting in an increased incidence and severity of respiratory tract infections. A recent Institute of Medicine study concluded that smoking increased the incidence of influenza and bacterial pneumonia and accounted for 19,000 smoking-related deaths per year (1). Children infected with Mycobacterium tuberculosis are five times more likely to develop pulmonary tuberculosis if exposed to cigarette smoke (2), and smoking doubles the risk of developing Pneumocystis carinii pneumonia in human immunodeficiency virus-infected individuals (3). Several studies have demonstrated that smoking suppresses T and B cell responses in the lungs without affecting cells in the peripheral blood (4 -7), but little research has been done to elucidate the underlying mechanism behind this phenomenon.We have recently identified two classes of immunosuppressive compounds in cigarette smoke. The dihydroxyphenols (hydroquinone and catechol) in the particulate phase inhibit T cell proliferation by blocking cell cycle progression in late G 1 and S phase (8 -12). In addition, the ␣,␤-unsaturated aldehydes, acrolein (CH 2 ACHCHO) and crotonaldehyde (CH 3 CHACHCHO) in the gas phase of cigarette smoke inhibit the production of several proinflammatory cytokines including IL-2, 4 tumor necrosis factor-␣, and granulocyte-macrophage colony-stimulating factor, with an IC 50 of 3 and 6 M, respectively (13). The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) have IC 50 values of Ͼ1500 M. The typical cigarett...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Acrolein Inhibits Cytokine Gene Expression by Alkylating Cysteine and Arginine Residues in the NF-κB1 DNA Binding Domain

Lambert¹,

Li²,

Jonscher

et al. 2007

Journal of Biological Chemistry

107

View full text Add to dashboard Cite

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“…The in vitro experiment also revealed acrolein adduction to Arg-307, forming six-membered heterocycle. From previous mutation studies in combination with thiol labeling approaches, it was known that Cys-61 is critical for the DNA-binding activity of p50 [103,104]. Analysis of the crystal structure of the NF-κB (p50/p65 heterodimer) bound to DNA supports a role of Arg-307 in mediating binding of p50 to DNA [105].…”

Section: Acrolein As a Modulator Of Stress-mediated Gene Activationmentioning

confidence: 96%

Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease

Stevens

Maier

2008

Molecular Nutrition Food Res

630

634

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Acrolein (2-propenal) is ubiquitously present in (cooked) foods and in the environment. It is formed from carbohydrates, vegetable oils and animal fats, amino acids during heating of foods, and by combustion of petroleum fuels and biodiesel. Chemical reactions responsible for release of acrolein include heat-induced dehydration of glycerol, retro-aldol cleavage of dehydrated carbohydrates, lipid peroxidation of polyunsaturated fatty acids, and Strecker degradation of methionine and threonine. Smoking of tobacco products equals or exceeds the total human exposure to acrolein from all other sources. The main endogenous sources of acrolein are myeloperoxidase-mediated degradation of threonine and amine oxidase-mediated degradation of spermine and spermidine, which may constitute a significant source of acrolein in situations of oxidative stress and inflammation. Acrolein is metabolized by conjugation with glutathione and excreted in the urine as mercapturic acid metabolites. Acrolein forms Michael adducts with ascorbic acid in vitro, but the biological relevance of this reaction is not clear. The biological effects of acrolein are a consequence of its reactivity towards biological nucleophiles such as guanine in DNA and cysteine, lysine, histidine, and arginine residues in critical regions of nuclear factors, proteases, and other proteins. Acrolein adduction disrupts the function of these biomacromolecules which may result in mutations, altered gene transcription, and modulation of apoptosis.

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“…However, there is evidence that ROS may also inhibit npg the NF-κB signaling pathway [46] . It has been reported that cysteine residue 179 in the activation loop of IKK-β [47,48] and that several conserved cysteine residues in the DNA-binding loop of the NF-κB/Rel proteins [49][50][51] may be targeted by ROS. P1 may inhibit NF-κB activation by modifying Cys-179 of IKK-β, as the inhibitory effects of P1 on NF-κB activity were abolished by the expression of mutant IKK-β, which contains alanine at residue 179 rather than cysteine ( Figure 5F).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-κB signaling pathway with distinct mechanisms

et al. 2013

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Aim: Curcumin has shown promising anticancer activity, which relies on its inhibition on NF-κB pathway. In this study, we characterized the pharmacological profile of a novel curcumin analog P1 and elucidate the related mechanisms. Methods: HEK293/NF-κB cells, stably transfected with an NF-κB-responsive luciferase reporter plasmid, were generated for highthroughput screen (HTS). Eight cancer cell lines, including PC3, COLO 205, HeLa cells etc. were tested. Cell viability was assessed using the sulforhodamine B (SRB) assays. Cell apoptosis was evaluated using FACS, immunocytochemistry, and Western blotting. H 2 -DCFDA and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS). The mitochondrial function was evaluated using mitochondrial oxygen consumption assay. Results: P1, a tropinone curcumin, was found in HTS targeting the NF-κB pathway. Its IC 50 value in inhibition of TNF-α-induced NF-κB activation was 0.8 μmol/L, whereas its IC 50 values in inhibiting the growth of A549 and HeLa cells were 1.24 and 0.69 μmol/L, respectively, which was 20-to 30-fold more potent than curcumin. The inhibition of P1 on the NF-κB pathway was further addressed in HeLa cells. The compound up to 10 µmol/L did not affect the binding of NF-κB to DNA, but markedly inhibited NF-κB nuclear translocation, IκB degradation and IκB kinase phosphorylation. The compound (1 and 3 µmol/L) concentration-dependently induced ROS generation, whereas curcumin up to 20 µmol/L had no effect. P1-induced ROS generation was mainly localized in mitochondria, and reversed by NAC. Moreover, the compound significantly enhanced TNF-α-induced apoptosis. Conclusion: P1 is a novel curcumin analog with potent anticancer activities, which exerts a distinct inhibition on the NF-κB pathway.

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Role of cysteine₆₂in DNA recognition by the P50 subunit of NF-_xB

Cited by 146 publications

References 32 publications

Acrolein Inhibits Cytokine Gene Expression by Alkylating Cysteine and Arginine Residues in the NF-κB1 DNA Binding Domain

Acrolein Inhibits Cytokine Gene Expression by Alkylating Cysteine and Arginine Residues in the NF-κB1 DNA Binding Domain

Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease

Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-κB signaling pathway with distinct mechanisms

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