I kappa B kinase beta (IKK beta) subunit of IKK complex is essential for the activation of NF-kappa B in response to various proinflammatory signals. Cys-179 in the activation loop of IKK beta is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKK beta in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKK beta activation was reduced in the IKK beta (C179A) mutant. The activity of IKK beta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappa B inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1(MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKK beta (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKK beta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.
Transfection ; Serine/*metabolism ; Protein Binding ; Phosphorylation ; Mutant Proteins/chemistry ; MAP Kinase Kinase Kinases/metabolism ; I-kappa B Kinase/*chemistry ; Humans ; Hela Cells ; Enzyme Activation/*physiology ; Cysteine/*physiology ; Cells, Cultured ; Catalytic Domain ; Amino Acid Substitution/physiology ; Adenosine Triphosphate/metabolism

The NF-kappaB pathway regulates the expression of over 150 target genes, e.g., cytokines, chemokines, leukocyte adhesion molecules and inducible effector enzymes. Consequently, it plays a crucial role in innate and adaptive immune responses, inflammatory response, stress responses, apoptosis and so on. IkappaB kinase (IKK) is the key of this pathway, and it owns a special structure which consists of catalytic subunit and regulatory subunit. Naturally, the activation of IKK needs the interaction of the two subunits and phosphorylation by its upstream kinases. Actually, there are two methods of activation of the NF-kappaB pathway, and both of the methods need the IKK complex. Given to the crucial role of IKK, researchers have isolated and synthesized amounts of IKK inhibitors, and these provide a great convenience to develop novel anti-inflammatory and anti-tumor drugs.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Antineoplastic Agents ; pharmacology ; Enzyme Activation ; Enzyme Inhibitors ; metabolism ; pharmacology ; Humans ; I-kappa B Kinase ; antagonists & inhibitors ; chemistry ; metabolism ; physiology ; NF-kappa B ; metabolism ; Phosphorylation ; Signal Transduction

Corn silk has been purified as an anticoagulant previously and the active component is a polysaccharide with a molecular mass of 135 kDa. It activates murine macrophages to induce nitric oxide synthase (NOS) and generate substantial amounts of NO in time and dose-dependent manners. It was detectable first at 15 h after stimulation by corn silk, peaked at 24 h, and undetectable by 48 h. Induction of NOS is inhibited by pyrolidine dithiocarbamate (PDTC) and genistein, an inhibitor of nuclear factor kappa B (NF-kappaB) and tyrosine kinase, respectively, indicating that iNOS stimulated by corn silk is associated with tyrosine kinase and NF-kappaB signaling pathways. IkappaB-alpha degradation was detectible at 10 min, and the level was restored at 120 min after treatment of corn silk. Corn silk induced nuclear translocation of NF-kappaB by phosphorylation and degradation of IkappaB-alpha.
Animals ; Anticoagulants/*pharmacology ; Genistein/pharmacology ; I-kappa B/metabolism ; Macrophages/drug effects/*enzymology/metabolism ; Mice ; NF-kappa B/antagonists & inhibitors/metabolism ; Nitric Oxide/biosynthesis ; Nitric-Oxide Synthase/antagonists & inhibitors/*biosynthesis ; Phosphorylation ; Plant Extracts/pharmacology ; Polysaccharides/*pharmacology ; Protein Transport/drug effects ; Protein-Tyrosine Kinase/antagonists & inhibitors/physiology ; Pyrrolidines/pharmacology ; Research Support, Non-U.S. Gov't ; Thiocarbamates/pharmacology ; Zea mays/*chemistry

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination

TANK-binding kinase 1 (TBK1) is an important enzyme in the regulation of cellular antiviral effects. TBK1 regulates the activity of the interferon regulatory factors IRF3 and IRF7, thereby playing a key role in type I interferon (IFN) signaling pathways. The structure of TBK1 consists of an N-terminal kinase domain, a middle ubiquitin-like domain (ULD), and a C-terminal elongated helical domain. It has been reported that the ULD of TBK1 regulates kinase activity, playing an important role in signaling and mediating interactions with other molecules in the IFN pathway. In this study, we present the crystal structure of the ULD of human TBK1 and identify several conserved residues by multiple sequence alignment. We found that a hydrophobic patch in TBK1, containing residues Leu316, Ile353, and Val382, corresponding to the "Ile44 hydrophobic patch" observed in ubiquitin, was conserved in TBK1, IκB kinase epsilon (IKKɛ/IKKi), IκB kinase alpha (IKKα), and IκB kinase beta (IKKβ). In comparison with the structure of the IKKβ ULD domain of Xenopus laevis, we speculate that the Ile44 hydrophobic patch of TBK1 is present in an intramolecular binding surface between ULD and the C-terminal elongated helices. The varying surface charge distributions in the ULD domains of IKK and IKK-related kinases may be relevant to their specificity for specific partners.
Amino Acid Sequence ; Conserved Sequence ; Crystallography, X-Ray ; Humans ; Hydrophobic and Hydrophilic Interactions ; I-kappa B Kinase ; chemistry ; metabolism ; Isoleucine ; chemistry ; Models, Molecular ; Molecular Sequence Data ; Protein Binding ; Protein Multimerization ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; chemistry ; Sequence Alignment ; Static Electricity ; Ubiquitin ; chemistry

This study is to investigate the protective effect of mangiferin on NF-kappaB (P65) and IkappaBalpha expression in peripheral blood mononuclear cell (PBMC) in rats with cigarette smoke induced chronic bronchitis. The rat model with chronic bronchitis was established by cigarette smoke. Real-time fluorescence RT-PCR was executed for evaluating the NF-kappaB (P65) and IKkappaBalpha gene expression in mononuclear cell, and flow cytometry for their protein expression. The serum hs-CRP (high-sensitivity C-reactive proteins) and TNF-alpha (tumor necrosis factor-alpha) were detected by enzyme-linked immunosorbent assay. The histopathological score was obtained from lung tissue HE staining slides of lung tissue. The results showed that mangiferin could markedly suppress the NF-kappaB (P65) mRNA and protein expression in mononuclear cell, while promote the IkappaBalpha mRNA and protein expression. Furthermore, mangiferin could lower serum hs-CRP and TNF-alpha level, and reduce the chronic inflammatory damage of bronchiole. These results suggested that mangiferin could notably ameliorate chronic bronchiole inflammation induced by cigarette smoke, and this protective effect might be linked to the regulation of NF-kappaB (P65) and IkappaBalpha expression in mononuclear cell.
Animals ; Bronchi ; pathology ; Bronchitis, Chronic ; blood ; etiology ; metabolism ; pathology ; C-Reactive Protein ; metabolism ; I-kappa B Kinase ; genetics ; metabolism ; Leukocytes, Mononuclear ; metabolism ; pathology ; Male ; Mangifera ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tobacco Smoke Pollution ; Transcription Factor RelA ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; Xanthones ; isolation & purification ; pharmacology

Microglial cells are the resident innate immune cells that sense pathogens and tissue injury in the central nervous system (CNS). Microglial activation is critical for neuroinflammatory responses. The synthetic compound 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139) is a novel chalcone-derived compound. In this study, we investigated the effects of DK-139 on Toll-like receptor 4 (TLR4)-mediated inflammatory responses in BV2 microglial cells. DK-139 inhibited lipopolysaccharide (LPS)-induced TLR4 activity, as determined using a cell-based assay. DK-139 blocked LPS-induced phosphorylation of IkappaB and p65/RelA NF-kappaB, resulting in inhibition of the nuclear translocation and trans-acting activity of NF-kappaB in BV2 microglial cells. We also found that DK-139 reduced the expression of NF-kappaB target genes, such as those for COX-2, iNOS, and IL-1beta, in LPS-stimulated BV2 microglial cells. Interestingly, DK-139 blocked LPS-induced Akt phosphorylation. Inhibition of Akt abrogated LPS-induced phosphorylation of p65/RelA, while overexpression of dominant-active p110CAAX enhanced p65/RelA phosphorylation as well as iNOS and COX2 expression. These results suggest that DK-139 exerts an anti-inflammatory effect on microglial cells by inhibiting the Akt/IkappaB kinase (IKK)/NF-kappaB signaling pathway.
Animals ; Binding Sites ; Cell Line ; Chalcones/chemistry/*pharmacology ; Cyclooxygenase 2/metabolism ; I-kappa B Kinase/metabolism ; Inflammation/*drug therapy ; Interleukin-1beta/metabolism ; Lipopolysaccharides/immunology ; Microglia/*drug effects/immunology/metabolism ; Molecular Dynamics Simulation ; NF-kappa B/*antagonists & inhibitors ; Nitric Oxide Synthase Type II/metabolism ; Phosphorylation/drug effects ; Protein Binding ; Proto-Oncogene Proteins c-akt/*antagonists & inhibitors ; Rats ; Signal Transduction ; Toll-Like Receptor 4/*antagonists & inhibitors/metabolism ; Transcription Factor RelA/metabolism

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.
1-Phosphatidylinositol 3-Kinase/metabolism ; Alkyl and Aryl Transferases/metabolism ; Animals ; Anticholesteremic Agents/pharmacology ; Cells, Cultured ; Enzyme Inhibitors/metabolism/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Gene Expression Regulation, Enzymologic/*drug effects ; I-kappa B Kinase/antagonists & inhibitors/metabolism ; Macrophages, Alveolar/cytology/*drug effects/*enzymology ; Matrix Metalloproteinase 9/genetics/*metabolism ; Mitogen-Activated Protein Kinase Kinases/metabolism ; Polyisoprenyl Phosphates/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Sesquiterpenes/metabolism ; Signal Transduction/physiology ; Simvastatin/*pharmacology ; Smoke/*adverse effects ; *Tobacco/adverse effects/chemistry

<b>OBJECTIVEb>To study the characteristics of lymphocyte nuclear factor kappa B (NF-&kappa;B) signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the intervening effect of Epimedium flavonoids (EF) on it.

<b>METHODSb>Sixty SD rats were divided into six groups, according to animals' age, i.e., the 3 days (d) group, the 4 months (m) group, the 10 m group, the 18 m group, the 27 m group, and the 27 m+EF group. RNA was extracted from separated splenic lymphocytes. Adopting NF-&kappa;B signal path functional genome oligonucleotide gene-chip (128 related genes), the integral characteristics and differences of NF-&kappa;B signal transduction kinase-related mRNA expressions were determined, and the intervening effect of EF was examined.

<b>RESULTSb>The mean level of the NF-&kappa;B signal transduction kinase-related mRNA expressions in rats' splenic lymphocytes lowered with aging; the highest expression was presented at 3 d after birth, and then, it lowered gradually, with the lowest level at 18 m or 27 m. After EF intervention, the expression level was raised to the 10-18 m level in the aged rats.

<b>CONCLUSIONb>The changing rules of lymphocyte NF-&kappa;B-signal-transduction-kinase-related mRNA expressions in various stages of aging are helpful for selecting the well time for preventing and intervening aging, and will also give a hint to the molecular index for assessment of senility retarding researches.

Aging ; drug effects ; genetics ; Animals ; Epimedium ; chemistry ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; I-kappa B Kinase ; metabolism ; Lymphocytes ; enzymology ; Male ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; genetics

Pentraxin 3 (PTX3) was identified as a marker of the inflammatory response and overexpressed in various tissues and cells related to cardiovascular disease. Honokiol, an active component isolated from the Chinese medicinal herb Magnolia officinalis, was shown to have a variety of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on palmitic acid (PA)-induced dysfunction of human umbilical vein endothelial cells (HUVECs) and to elucidate potential regulatory mechanisms in this atherosclerotic cell model. Our results showed that PA significantly accelerated the expression of PTX3 in HUVECs through the IkappaB kinase (IKK)/IkappaB/nuclear factor-kappaB (NF-kappaB) pathway, reduced cell viability, induced cell apoptosis and triggered the inflammatory response. Knockdown of PTX3 supported cell growth and prevented apoptosis by blocking PA-inducted nitric oxide (NO) overproduction. Honokiol significantly suppressed the overexpression of PTX3 in PA-inducted HUVECs by inhibiting IkappaB phosphorylation and the expression of two NF-kappaB subunits (p50 and p65) in the IKK/IkappaB/NF-kappaB signaling pathway. Furthermore, honokiol reduced endothelial cell injury and apoptosis by regulating the expression of inducible NO synthase and endothelial NO synthase, as well as the generation of NO. Honokiol showed an anti-inflammatory effect in PA-inducted HUVECs by significantly inhibiting the generation of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1. In summary, honokiol repaired endothelial dysfunction by suppressing PTX3 overexpression in an atherosclerotic cell model. PTX3 may be a potential therapeutic target for atherosclerosis.
Apoptosis/drug effects ; Atherosclerosis/chemically induced/*drug therapy/immunology/pathology ; Biphenyl Compounds/chemistry/isolation & purification/*pharmacology ; C-Reactive Protein/*genetics/immunology ; Down-Regulation/drug effects ; Drugs, Chinese Herbal/chemistry/isolation & purification/*pharmacology ; Human Umbilical Vein Endothelial Cells ; Humans ; I-kappa B Kinase/*immunology ; Lignans/chemistry/isolation & purification/*pharmacology ; Magnolia/chemistry ; Palmitic Acid ; Protein-Serine-Threonine Kinases/*immunology ; Serum Amyloid P-Component/*genetics/immunology ; Signal Transduction/drug effects