Sample records for proteins activate factor
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Factor VII and protein C are phosphatidic acid-binding proteins.
Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H
2013-08-20
Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.
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NASA Technical Reports Server (NTRS)
Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.
2000-01-01
Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.
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Cai, Min; Tong, Li; Dong, Beibei; Hou, Wugang; Shi, Likai; Dong, Hailong
2017-03-01
The authors have reported that antioxidative effects play a crucial role in the volatile anesthetic-induced neuroprotection. Accumulated evidence shows that endogenous antioxidation could be up-regulated by nuclear factor-E2-related factor 2 through multiple pathways. However, whether nuclear factor-E2-related factor 2 activation is modulated by sevoflurane preconditioning and, if so, what is the signaling cascade underlying upstream of this activation are still unknown. Sevoflurane preconditioning in mice was performed with sevoflurane (2.5%) 1 h per day for five consecutive days. Focal cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion. Expression of nuclear factor-E2-related factor 2, kelch-like ECH-associated protein 1, manganese superoxide dismutase, thioredoxin-1, and nicotinamide adenine dinucleotide phosphate quinolone oxidoreductase-1 was detected (n = 6). The antioxidant activities and oxidative product expression were also examined. To determine the role of kelch-like ECH-associated protein 1 inhibition-dependent nuclear factor-E2-related factor 2 activation in sevoflurane preconditioning-induced neuroprotection, the kelch-like ECH-associated protein 1-nuclear factor-E2-related factor 2 signal was modulated by nuclear factor-E2-related factor 2 knockout, kelch-like ECH-associated protein 1 overexpression lentivirus, and kelch-like ECH-associated protein 1 deficiency small interfering RNA (n = 8). The infarct volume, neurologic scores, and cellular apoptosis were assessed. Sevoflurane preconditioning elicited neuroprotection and increased nuclear factor-E2-related factor 2 nuclear translocation, which in turn up-regulated endogenous antioxidation and reduced oxidative injury. Sevoflurane preconditioning reduced kelch-like ECH-associated protein 1 expression. Nuclear factor-E2-related factor 2 ablation abolished neuroprotection and reversed sevoflurane preconditioning by mediating the up-regulation of antioxidants. Kelch-like ECH-associated protein 1 overexpression reversed nuclear factor-E2-related factor 2 up-regulation and abolished the neuroprotection induced by sevoflurane preconditioning. Kelch-like ECH-associated protein 1 small interfering RNA administration improved nuclear factor-E2-related factor 2 expression and the outcome of mice subjected to ischemia/reperfusion injury. Kelch-like ECH-associated protein 1 down-regulation-dependent nuclear factor-E2-related factor 2 activation underlies the ability of sevoflurane preconditioning to activate the endogenous antioxidant response, which elicits its neuroprotection.
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Chen, Hsiu-Hui; Vicente, Cristina P.; He, Li; Tollefsen, Douglas M.; Wun, Tze-Chein
2005-01-01
The anionic phospholipid, phosphatidyl-l-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid β-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor–initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents. PMID:15677561
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Ruiz-Torres, M P; Perez-Rivero, G; Diez-Marques, M L; Griera, M; Ortega, R; Rodriguez-Puyol, M; Rodríguez-Puyol, D
2007-01-01
While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-beta1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-beta1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-beta1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-beta1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.
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Complement factor H family proteins in their non-canonical role as modulators of cellular functions.
Józsi, Mihály; Schneider, Andrea E; Kárpáti, Éva; Sándor, Noémi
2018-01-04
Complement factor H is a major regulator of the alternative pathway of the complement system. The factor H-related proteins are less characterized, but recent data indicate that they rather promote complement activation. These proteins have some common ligands with factor H and have both overlapping and distinct functions depending on domain composition and the degree of conservation of amino acid sequence. Factor H and some of the factor H-related proteins also appear in a non-canonical function that is beyond their role in the modulation of complement activation. This review covers our current understanding on this emerging role of factor H family proteins in modulating the activation and function of various cells by binding to receptors or receptor ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H
1997-03-01
A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.
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Binding Mode Analysis of Zerumbone to Key Signal Proteins in the Tumor Necrosis Factor Pathway
Fatima, Ayesha; Abdul, Ahmad Bustamam Hj.; Abdullah, Rasedee; Karjiban, Roghayeh Abedi; Lee, Vannajan Sanghiran
2015-01-01
Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis. PMID:25629232
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[Regulation of heat shock gene expression in response to stress].
Garbuz, D G
2017-01-01
Heat shock (HS) genes, or stress genes, code for a number of proteins that collectively form the most ancient and universal stress defense system. The system determines the cell capability of adaptation to various adverse factors and performs a variety of auxiliary functions in normal physiological conditions. Common stress factors, such as higher temperatures, hypoxia, heavy metals, and others, suppress transcription and translation for the majority of genes, while HS genes are upregulated. Transcription of HS genes is controlled by transcription factors of the HS factor (HSF) family. Certain HSFs are activated on exposure to higher temperatures or other adverse factors to ensure stress-induced HS gene expression, while other HSFs are specifically activated at particular developmental stages. The regulation of the main mammalian stress-inducible factor HSF1 and Drosophila melanogaster HSF includes many components, such as a variety of early warning signals indicative of abnormal cell activity (e.g., increases in intracellular ceramide, cytosolic calcium ions, or partly denatured proteins); protein kinases, which phosphorylate HSFs at various Ser residues; acetyltransferases; and regulatory proteins, such as SUMO and HSBP1. Transcription factors other than HSFs are also involved in activating HS gene transcription; the set includes D. melanogaster GAF, mammalian Sp1 and NF-Y, and other factors. Transcription of several stress genes coding for molecular chaperones of the glucose-regulated protein (GRP) family is predominantly regulated by another stress-detecting system, which is known as the unfolded protein response (UPR) system and is activated in response to massive protein misfolding in the endoplasmic reticulum and mitochondrial matrix. A translational fine tuning of HS protein expression occurs via changing the phosphorylation status of several proteins involved in translation initiation. In addition, specific signal sequences in the 5'-UTRs of some HS protein mRNAs ensure their preferential translation in stress.
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Yamamoto, N
1996-10-01
Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.
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Molecular switch-like regulation in motor proteins.
Tafoya, Sara; Bustamante, Carlos
2018-06-19
Motor proteins are powered by nucleotide hydrolysis and exert mechanical work to carry out many fundamental biological tasks. To ensure their correct and efficient performance, the motors' activities are allosterically regulated by additional factors that enhance or suppress their NTPase activity. Here, we review two highly conserved mechanisms of ATP hydrolysis activation and repression operating in motor proteins-the glutamate switch and the arginine finger-and their associated regulatory factors. We examine the implications of these regulatory mechanisms in proteins that are formed by multiple ATPase subunits. We argue that the regulatory mechanisms employed by motor proteins display features similar to those described in small GTPases, which require external regulatory elements, such as dissociation inhibitors, exchange factors and activating proteins, to switch the protein's function 'on' and 'off'. Likewise, similar regulatory roles are taken on by the motor's substrate, additional binding factors, and even adjacent subunits in multimeric complexes. However, in motor proteins, more than one regulatory factor and the two mechanisms described here often underlie the machine's operation. Furthermore, ATPase regulation takes place throughout the motor's cycle, which enables a more complex function than the binary 'active' and 'inactive' states.This article is part of a discussion meeting issue 'Allostery and molecular machines'. © 2018 The Author(s).
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Dosil, Mercedes; Schandel, Kimberly A.; Gupta, Ekta; Jenness, Duane D.; Konopka, James B.
2000-01-01
Binding of the α-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Gα subunits in an α-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Gα subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the α-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for α-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the α-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor–G-protein preactivation complexes. PMID:10866688
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Thrombophilic mutations in pre-eclampsia and pregnancy-induced hypertension.
Omar, Siti Z; Qvist, Rajes; Khaing, Si L; Muniandy, Sekaran; Bhalla, Sunil
2008-04-01
The aim of the present study was to determine the existence or prevalence of thrombophilic markers such as Factor V Leiden, prothrombin G20210A, protein S, protein C, activated protein C and anti-thrombin in pre-eclampsia and pregnancy-induced hypertensive patients. Blood samples were collected from a total number of 124 women at the maternity unit, University of Malaya Medical Center. These included 49 patients with pre-eclampsia, 63 patients with pregnancy-induced hypertension and 12 normal pregnant women. DNA was extracted from the blood samples. Factor V Leiden (Taq I) and prothrombin G20210A (Hind III) genotyping was done on polymerase chain reaction-restriction fragment length polymorphism. Anti-thrombin activity and the concentrations of protein C, protein S and activated protein C were measured using the IL Coagulation System (Hemosil). Of the 124 subjects, one pre-eclampsia patient was homozygous for Factor V Leiden mutation but prothrombin G20210A mutation was not present in any of the subjects. The subject with Factor V Leiden mutation also had a low activated protein C resistance and a low protein S concentration. Factor V Leiden mutation is present in the Asian population and may very well serve as one of the genetic factors responsible for pre-eclampsia and other adverse pregnancy outcomes.
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Weinstein, M J; Chute, L E
1984-01-01
We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity. Images PMID:6421875
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The sigma factors of Bacillus subtilis.
Haldenwang, W G
1995-01-01
The specificity of DNA-dependent RNA polymerase for target promotes is largely due to the replaceable sigma subunit that it carries. Multiple sigma proteins, each conferring a unique promoter preference on RNA polymerase, are likely to be present in all bacteria; however, their abundance and diversity have been best characterized in Bacillus subtilis, the bacterium in which multiple sigma factors were first discovered. The 10 sigma factors thus far identified in B. subtilis directly contribute to the bacterium's ability to control gene expression. These proteins are not merely necessary for the expression of those operons whose promoters they recognize; in many instances, their appearance within the cell is sufficient to activate these operons. This review describes the discovery of each of the known B. subtilis sigma factors, their characteristics, the regulons they direct, and the complex restrictions placed on their synthesis and activities. These controls include the anticipated transcriptional regulation that modulates the expression of the sigma factor structural genes but, in the case of several of the B. subtilis sigma factors, go beyond this, adding novel posttranslational restraints on sigma factor activity. Two of the sigma factors (sigma E and sigma K) are, for example, synthesized as inactive precursor proteins. Their activities are kept in check by "pro-protein" sequences which are cleaved from the precursor molecules in response to intercellular cues. Other sigma factors (sigma B, sigma F, and sigma G) are inhibited by "anti-sigma factor" proteins that sequester them into complexes which block their ability to form RNA polymerase holoenzymes. The anti-sigma factors are, in turn, opposed by additional proteins which participate in the sigma factors' release. The devices used to control sigma factor activity in B, subtilis may prove to be as widespread as multiple sigma factors themselves, providing ways of coupling sigma factor activation to environmental or physiological signals that cannot be readily joined to other regulatory mechanisms. PMID:7708009
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Abe, Cecília M.; Monaris, Denize; Morais, Zenaide M.; Souza, Gisele O.; Vasconcellos, Sílvio A.; Isaac, Lourdes; Abreu, Patrícia A. E.; Barbosa, Angela S.
2013-01-01
The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities. PMID:24312361
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Adams, David S; Hasson, Brendan; Boyer-Boiteau, Anne; El-Khishin, Adam; Shashoua, Victor E
2003-05-01
Ependymin (EPN) is a goldfish brain neurotrophic factor previously shown to function in a variety of cellular events related to long-term memory formation and neuronal regeneration. CMX-8933, an 8-amino-acid synthetic peptide fragment of EPN, was designed for aiding an investigation of the biological properties of this glycoprotein. We reported from previous studies that treatment of mouse neuroblastoma (NB2a) cultures with CMX-8933 promotes activation of transcription factor AP-1, a characteristic previously associated with the following full-length neurotrophic factors: nerve growth factor, neurotropin-3, and brain-derived neurotrophic factor. The CMX-8933-activated AP-1 specifically bound an AP-1 consensus probe and appeared to contain c-Jun and c-Fos protein components in antibody supershift experiments. Because AP-1 influences a variety of positive and negative cellular processes, determined in part by its exact protein composition and mechanism of activation, we extended these initial AP-1 observations in the current study to confirm the identity of the CMX-8933-activated c-Jun and c-Fos components. CMX-8933 increases the enzymatic activity of c-Jun N-terminal kinase (JNK), increases the phosphorylation of JNK and c-Jun proteins, and increases the cellular titers of c-Jun and c-Fos mRNAs. Furthermore, the AP-1 activated by CMX-8933 is functional, insofar as it transactivates both synthetic and natural AP-1-dependent reporter plasmids. Inhibition studies indicate that activation of the 8933-induced AP-1 occurs via the mitogen-activated protein kinase pathway. These data are in agreement with the recently proposed model for the conversion of short- to long-term synaptic plasticity and memory, in which a JNK-activated transcription factor AP-1, containing c-Jun and c-Fos components, functions at the top of a hierarchy of transcription factors known to regulate long-term neural plasticity. Copyright 2003 Wiley-Liss, Inc.
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The activation of plasminogen by Hageman factor (Factor XII) and Hageman factor fragments.
Goldsmith, G H; Saito, H; Ratnoff, O S
1978-01-01
Activation of plasminogen through surface-mediated reactions is well recognized. In the presence of kaolin, purified Hageman factor (Factor XII) changed plasminogen to plasmin, as assayed upon a synthetic amide substrate and by fibrinolysis. Kinetic studies suggested an enzymatic action of Hageman factor upon its substrate, plasminogen. Hageman factor fragments, at a protein concentration equivalent to whole Hageman factor, activated plasminogen to a lesser extent. These protein preparations were not contaminated with other agents implicated in surface-mediated fibrinolysis. Diisopropyl fluorophosphate treatment of plasminogen did not inhibit its activation by Hageman factor. These studies indicate that Hageman factor has a hitherto unsuspected function, the direct activation of plasminogen. PMID:659637
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Gao, Song; Carson, James A
2016-01-01
Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. Copyright © 2016 the American Physiological Society.
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Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe
2012-03-01
Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2). Copyright © 2011 Elsevier Ltd. All rights reserved.
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Venom Protein C activators as diagnostic agents for defects of protein C System.
Ramzan, Faiqah; Asmat, Andleeb
2018-06-18
Background Protein C is a vitamin K dependent plasma zymogen. It prevents clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converts the zymogen Protein C (PC) of vertebrates into activated PC, which has been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques. Conclusion The fast-acting PC activator Protac® from Agkistrodon contortrix (southern copperhead snake) venom has been found to have broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Identification of a novel A20-binding inhibitor of nuclear factor-kappa B activation termed ABIN-2.
Van Huffel, S; Delaei, F; Heyninck, K; De Valck, D; Beyaert, R
2001-08-10
The nuclear factor kappaB (NF-kappaB) plays a central role in the regulation of genes implicated in immune responses, inflammatory processes, and apoptotic cell death. The zinc finger protein A20 is a cellular inhibitor of NF-kappaB activation by various stimuli and plays a critical role in terminating NF-kappaB responses. The underlying mechanism for NF-kappaB inhibition by A20 is still unknown. A20 has been shown to interact with several proteins including tumor necrosis factor (TNF) receptor-associated factors 2 and 6, as well as the inhibitory protein of kappaB kinase (IKK) gamma protein. Here we report the cloning and characterization of ABIN-2, a previously unknown protein that binds to the COOH-terminal zinc finger domain of A20. NF-kappaB activation induced by TNF and interleukin-1 is inhibited by overexpression of ABIN-2. The latter also inhibits NF-kappaB activation induced by overexpression of receptor-interacting protein or TNF receptor-associated factor 2. In contrast, NF-kappaB activation by overexpression of IKKbeta or direct activators of the IKK complex, such as Tax, cannot be inhibited by ABIN-2. These results indicate that ABIN-2 interferes with NF-kappaB activation upstream of the IKK complex and that it might contribute to the NF-kappaB-inhibitory function of A20.
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Human Protein Kinases and Obesity.
Engin, Atilla
2017-01-01
The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified by the target amino acid in their substrates. Some protein kinases can phosphorylate both serine/threonine, as well as tyrosine residues. This group of kinases has been known as dual specificity kinases. Unlike the dual specificity kinases, a heterogeneous group of protein phosphatases are known as dual-specificity phosphatases. These phosphatases remove phosphate groups from tyrosine and serine/threonine residues on their substrate. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases. The protein kinase-phosphoproteins interactions play an important role in obesity . In obesity, the pro- and anti-inflammatory effects of adipokines and cytokines through intracellular signaling pathways mainly involve the nuclear factor kappa B (NF-kappaB) and the c-Jun N-terminal kinase (JNK) systems as well as the inhibitor of kappaB-kinase beta (IKK beta). Impairment of insulin signaling in obesity is largely mediated by the activation of the IKKbeta and the JNK. Furthermore, oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2alpha kinase (PERK) and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. Increase in intracellular oxidative stress can promote PKC-beta activation. Activated PKC-beta induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhances triglyceride accumulation. Obesity is fundamentally caused by cellular energy imbalance and dysregulation. Like adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), N-terminal Per-ARNT-Sim (PAS) kinase are nutrient responsive protein kinases and important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular level. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.
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Józsi, Mihály; Meri, Seppo
2014-01-01
Factor H-related proteins (CFHRs) are plasma glycoproteins related in structure and antigenicity to each other and to the complement inhibitory protein factor H. Such proteins are found in most mammals but their number and domain composition vary. This chapter summarizes our current knowledge on the human factor H-related proteins. In contrast to factor H, they have no strong complement inhibitory activity, although for some of them regulatory or complement modulatory activity has been reported. A common feature of CFHRs is that they bind to the C3b component of complement. Novel links between CFHRs and various diseases (C3 glomerulopathies, atypical hemolytic uremic syndrome and age-related macular degeneration) have been revealed in recent years, but we are still far from understanding their biological function.
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Requirement of GM2 ganglioside activator for phospholipase D activation
Nakamura, Shun-ichi; Akisue, Toshihiro; Jinnai, Hitoshi; Hitomi, Tomohiro; Sarkar, Sukumar; Miwa, Noriko; Okada, Taro; Yoshida, Kimihisa; Kuroda, Shun’ichi; Kikkawa, Ushio; Nishizuka, Yasutomi
1998-01-01
Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by β-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792–815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown. PMID:9770472
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Sun, Hongliu; Nelms, Brian L; Sleiman, Sama F; Chamberlin, Helen M; Hanna-Rose, Wendy
2007-10-01
The previously reported negative regulatory activity of HIM-8 on the Sox protein EGL-13 is shared by the HIM-8-related ZIM proteins. Furthermore, mutation of HIM-8 can modulate the effects of substitution mutations in the DNA-binding domains of at least four other transcription factors, suggesting broad regulatory activity by HIM-8.
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Alvarez-Flores, Miryam Paola; Furlin, Daniel; Ramos, Oscar H. P.; Balan, Andrea; Konno, Katsuhiro; Chudzinski-Tavassi, Ana Marisa
2011-01-01
Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X. PMID:21177860
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Jung, Gyung Ah; Shin, Bong Shik; Jang, Yeon Sue; Sohn, Jae Bum; Woo, Seon Rang; Kim, Jung Eun; Choi, Go; Lee, Kyung-Mi; Min, Bon Hong
2011-01-01
Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21Cip/WAF1 activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway. PMID:21778808
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Role of platelet activating factor in pathogenesis of acute pancreatitis in rats.
Konturek, S J; Dembinski, A; Konturek, P J; Warzecha, Z; Jaworek, J; Gustaw, P; Tomaszewska, R; Stachura, J
1992-01-01
The importance of platelet activating factor in acute pancreatitis was examined by determining the tissue content of endogenous platelet activating factor and the protective effects of TCV-309, a highly selective platelet activating factor blocker, against caerulein induced pancreatitis in rats. Infusion of caerulein (10 micrograms/kg/h) for five hours resulted in about 70% increase in pancreatic weight, 22% rise in protein content, 50% reduction in tissue blood flow, nine fold increase in tissue level of platelet activating factor and 165% rise in plasma amylase as well as histological evidence of acute pancreatitis. Such infusion of caerulein in chronic pancreatic fistula rats caused a marked increase in protein output from basal secretion of 10 mg/30 minutes to 40 mg/30 minutes in the first hour of infusion followed by a decline in protein output to 15-20 mg/30 minutes in the following hours of the experiment. Exogenous platelet activating factor (50 micrograms/kg) injected ip produced similar alterations in weight, protein content, blood flow, and histology of the pancreas but the increment in serum amylase was significantly smaller and pancreatic secretion was reduced below the basal level. TCV-309 (50 micrograms/kg) given ip before caerulein or platelet activating factor administration significantly reduced the biochemical and morphological alterations caused by caerulein and abolished those induced by exogenous platelet activating factor. These results indicate that platelet activating factor plays an important role in the pathogenesis of acute pancreatitis probably by reducing the blood flow and increasing vascular permeability in the pancreas. PMID:1385272
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Polyphenol Compound as a Transcription Factor Inhibitor.
Park, Seyeon
2015-10-30
A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)).
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Kuta, Piotr; Hauck-Dlimi, Barbara; Strobel, Julian; Zimmermann, Robert; Eckstein, Reinhold
2016-01-01
Uncontrolled hemorrhage in polytrauma patients usually results in rapid need of blood products. Despite the shorter thawing times of microwave devices for heating fresh frozen plasma (FFP), their use has remained controversial, and just a few laboratory analyses have been published on this topic. The aim of this study was to analyse the quality of clotting factors immediately after thawing FFP with a microwave device and after 48-hour post thaw storage at 4 degrees C. 24 FFP units of all four ABO blood groups (six of each blood group) were thawed with a Transfusio-therm 2000 and later stored at 4 degrees C for 48 hours. Samples were drawn aseptically and investigated on various clotting factors and protein proteases (fibrinogen, antithrombin, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, vWF antigen and activity, protein S, and protein C) using standard coagulation and chromogenic assays immediately after thawing and again after a 48-hour storage period at 4 degrees C. All units were tested for both anaerobic and aerobic microbial contamination using standard operating procedures immediately after thawing. After thawing, all coagulation factors and protein protease activities were within normal ranges. Blood group O individuals had approximately 25% lower plasma levels of vWF antigen and activity. After a 48-hour storage period at 4 degrees C, FVIII and FIX activities declined significantly in all blood groups, whereas the remaining clotting factors remained comparably stable. Immediately after rapid thawing using a microwave system, all FFP units contained adequate coagulation factor activities to maintain hemostatic activity at the time of product thaw. The post thaw refrigerated storage caused an anticipated decrease in factor VIII and IX activities, but retained normal coagulation factor levels of many plasma proteins. Therefore we conclude that the Transfusio-therm 2000 has no clinically significant influence on the activity of clotting factors and plasma proteases in FFP units.
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Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki; Ushijima, Naofumi
2008-01-15
Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized beta-galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years. Copyright 2007 Wiley-Liss, Inc.
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Mincheva, Stefka; Garcera, Ana; Gou-Fabregas, Myriam; Encinas, Mario; Dolcet, Xavier; Soler, Rosa M
2011-04-27
In vivo and in vitro motoneuron survival depends on the support of neurotrophic factors. These factors activate signaling pathways related to cell survival or inactivate proteins involved in neuronal death. In the present work, we analyzed the involvement of the nuclear factor-κB (NF-κB) pathway in mediating mouse spinal cord motoneuron survival promoted by neurotrophic factors. This pathway comprises ubiquitously expressed transcription factors that could be activated by two different routes: the canonical pathway, associated with IKKα/IKKβ kinase phosphorylation and nuclear translocation RelA (p65)/p50 transcription factors; and the noncanonical pathway, related to IKKα kinase homodimer phosphorylation and RelB/p52 transcription factor activation. In our system, we show that neurotrophic factors treatment induced IKKα and IKKβ phosphorylation and RelA nuclear translocation, suggesting NF-κB pathway activation. Protein levels of different members of the canonical or noncanonical pathways were reduced in a primary culture of isolated embryonic motoneurons using an interference RNA approach. Even in the presence of neurotrophic factors, selective reduction of IKKα, IKKβ, or RelA proteins induced cell death. In contrast, RelB protein reduction did not have a negative effect on motoneuron survival. Together these results demonstrated that the canonical NF-κB pathway mediates motoneuron survival induced by neurotrophic factors, and the noncanonical pathway is not related to this survival effect. Canonical NF-κB blockade induced an increase of Bim protein level and apoptotic cell death. Bcl-x(L) overexpression or Bax reduction counteracted this apoptotic effect. Finally, RelA knockdown causes changes of CREB and Smn protein levels.
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[CNC proteins in physiology and pathology].
Gęgotek, Agnieszka; Skrzydlewska, Elżbieta
2015-07-06
CNC proteins consist of Bach1, Bach2 and 4 homologous transcription factors: Nrf1, Nrf2, Nrf3 and p45NF-E2. Transcription factors belonging to this group of proteins play a crucial role in protection of cells against oxidative stress. Under physiological conditions, they remain in the cytoplasm in the inactive form or are degraded. However, in oxidative stress conditions, they are translocated to the nucleus, and bind to DNA in the ARE sequence. Consequently, there is transcription of genes encoding cytoprotective proteins, such as phase II enzymes, or low molecular weight antioxidant proteins (i.e., thioredoxin, ferritin, metallothionein) responsible for protecting cells from reactive oxygen species (ROS) action. The activity of transcriptional proteins depends directly on the redox state of the cell. ROS as second messenger signals, control inhibitors of cytoplasmic CNC proteins or potentiate the activity of kinases (MAPK, PKC, PI3K, PERK), leading to phosphorylation of transcription factors. This is conducive to translocation of these molecules into the nucleus and to formation of complexes that initiate the gene expression. Disorders of regulation of the activity of transcription factors belonging to the CNC proteins caused by gene mutations, epigenetic modifications or increased activity of p62, p21, or k-Ras, B-Raf and c-Myc oncogenes, induce changes in the level of ARE-dependent gene expression, which can lead even to the development of carcinogenesis. On the other hand, Nrf transcription factors, inducing the expression of antioxidants and enzymes responsible for the detoxification of xenobiotics, can be considered as a potential target of the action of chemopreventive factors in anticancer therapy.
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Hypoxia in Invasion and Metastasis
2007-08-01
hypoxia and activating HIF-1 downregulate the DNA mismatch repair proteins ( mlh1 and/or msh2), a group of important proteins for maintaining genetic...Investigate the hypoxia and activating HIF-1 downregulate the DNA mismatch repair proteins ( mlh1 and/or msh2) (Month 7-12) Methods: We performed a parallel...inducible factors from invasive tumor cells. Changes in the level of multiple hypoxia related factor (HIF-1) and DNA mismatch repair proteins ( MLH1 , MSH2
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Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF).
Yamamoto, Nobuto; Ushijima, Naofumi; Koga, Yoshihiko
2009-01-01
Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.
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Oocyte Activation and Fertilisation: Crucial Contributors from the Sperm and Oocyte.
Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Coward, Kevin
2017-01-01
This chapter intends to summarise the importance of sperm- and oocyte-derived factors in the processes of sperm-oocyte binding and oocyte activation. First, we describe the initial interaction between sperm and the zona pellucida, with particular regard to acrosome exocytosis. We then describe how sperm and oocyte membranes fuse, with special reference to the discovery of the sperm protein IZUMO1 and its interaction with the oocyte membrane receptor JUNO. We then focus specifically upon oocyte activation, the fundamental process by which the oocyte is alleviated from metaphase II arrest by a sperm-soluble factor. The identity of this sperm factor has been the source of much debate recently, although mounting evidence, from several different laboratories, provides strong support for phospholipase C ζ (PLCζ), a sperm-specific phospholipase. Herein, we discuss the evidence in support of PLCζ and evaluate the potential role of other candidate proteins, such as post-acrosomal WW-binding domain protein (PAWP/WBP2NL). Since the cascade of downstream events triggered by the sperm-borne oocyte activation factor heavily relies upon specialised cellular machinery within the oocyte, we also discuss the critical role of oocyte-borne factors, such as the inositol trisphosphate receptor (IP 3 R), protein kinase C (PKC), store-operated calcium entry (SOCE) and calcium/calmodulin-dependent protein kinase II (CaMKII), during the process of oocyte activation. In order to place the implications of these various factors and processes into a clinical context, we proceed to describe their potential association with oocyte activation failure and discuss how clinical techniques such as the in vitro maturation of oocytes may affect oocyte activation ability. Finally, we contemplate the role of artificial oocyte activating agents in the clinical rescue of oocyte activation deficiency and discuss options for more endogenous alternatives.
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Yamada, Kana; Noguchi, Chisato; Kamitori, Kazuyo; Dong, Youyi; Hirata, Yuko; Hossain, Mohammad A; Tsukamoto, Ikuko; Tokuda, Masaaki; Yamaguchi, Fuminori
2012-02-01
Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function. Copyright © 2012 Elsevier Inc. All rights reserved.
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The activity of the acidic phosphoproteins from the 80 S rat liver ribosome.
MacConnell, W P; Kaplan, N O
1982-05-25
The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes.
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Smith, M R; Greene, W C
1991-01-01
The Tax oncoprotein of the type I human T cell leukemia virus (HTLV-I) activates transcription of cellular and viral genes through at least two different transcription factor pathways. Tax activates transcription of the c-fos proto-oncogene by a mechanism that appears to involve members of the cAMP response element binding protein (CREB) and activating transcription factor (ATF) family of DNA-binding proteins. Tax also induces the nuclear expression of the NF-kappa B family of rel oncogene-related enhancer-binding proteins. We have investigated the potential role of these CREB/ATF and NF-kappa B/Rel transcription factors in Tax-mediated transformation by analyzing the oncogenic potential of Tax mutants that functionally segregate these two pathways of transactivation. Rat fibroblasts (Rat2) stably expressing either the wild-type Tax protein or a Tax mutant selectively deficient in the ability to induce NF-kappa B/Rel demonstrated marked changes in morphology and growth characteristics including the ability to form tumors in athymic mice. In contrast, Rat2 cells stably expressing a Tax mutant selectively deficient in the ability to activate transcription through CREB/ATF demonstrated no detectable changes in morphology or growth characteristics. These results suggest that transcriptional activation through the CREB/ATF pathway may play an important role in Tax-mediated cellular transformation. Images PMID:1832173
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Mohamad, Saharuddin Bin; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi
2002-01-01
Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF. Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity. Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.
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Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.
Li, Hongyan; Jeong, Hyung Min; Choi, You Hee; Lee, Sung Ho; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl
2013-05-10
Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix. Copyright © 2013 Elsevier Inc. All rights reserved.
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Huang, Lei; Kondo, Fumio; Harato, Misako; Feng, Guo-Gang; Ishikawa, Naoshisa; Fujiwara, Yoshihiro; Okada, Shoshiro
2014-01-01
The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression. PMID:24466034
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Rider, Mark H; Hussain, Nusrat; Dilworth, Stephen M; Storey, Kenneth B
2009-12-01
Long-term survival of oxygen deprivation by animals with well-developed anoxia tolerance depends on multiple biochemical adaptations including strong metabolic rate depression. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the suppression of protein synthesis that occurs when turtles experience anoxic conditions. AMPK activity and the phosphorylation state of ribosomal translation factors were measured in liver, heart, red muscle and white muscle of red-eared slider turtles (Trachemys scripta elegans) subjected to 20 h of anoxic submergence. AMPK activity increased twofold in white muscle of anoxic turtles compared with aerobic controls but remained unchanged in liver and red muscle, whereas in heart AMPK activity decreased by 40%. Immunoblotting with phospho-specific antibodies revealed that eukaryotic elongation factor-2 phosphorylation at the inactivating Thr56 site increased six- and eightfold in red and white muscles from anoxic animals, respectively, but was unchanged in liver and heart. The phosphorylation state of the activating Thr389 site of p70 ribosomal protein S6 kinase was reduced under anoxia in red muscle and heart but was unaffected in liver and white muscle. Exposure to anoxia decreased 40S ribosomal protein S6 phosphorylation in heart and promoted eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) dephosphorylation in red muscle, but surprisingly increased 4E-BP1 phosphorylation in white muscle. The changes in phosphorylation state of translation factors suggest that organ-specific patterns of signalling and response are involved in achieving the anoxia-induced suppression of protein synthesis in turtles.
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Nutrition and physical activity for the prevention and treatment of age-related sarcopenia.
Bosaeus, Ingvar; Rothenberg, Elisabet
2016-05-01
Sarcopenia, defined as loss of skeletal muscle mass and function, is associated with adverse outcomes such as physical disability, impaired quality of life and increased mortality. Several mechanisms are involved in the development of sarcopenia. Potentially modifiable factors include nutrition and physical activity. Protein metabolism is central to the nutritional issues, along with other potentially modifying nutritional factors as energy balance and vitamin D status. An increasing but still incomplete knowledge base has generated recent recommendations on an increased protein intake in the elderly. Several factors beyond the total amount of protein consumed emerge as potentially important in this context. A recent summit examined three hypotheses: (1) A meal threshold; habitually consuming 25-30 g protein at breakfast, lunch and dinner provides sufficient protein to effectively stimulate muscle protein anabolism; (2) Protein quality; including high-quality protein at each meal improves postprandial muscle protein synthesis; and (3) performing physical activity in close temporal proximity to a high-quality protein meal enhances muscle anabolism. Optimising the potential for muscle protein anabolism by consuming an adequate amount of high-quality protein at each meal, in combination with physical activity, appears as a promising strategy to prevent or delay the onset of sarcopenia. However, results of interventions are inconsistent, and well-designed, standardised studies evaluating exercise or nutrition interventions are needed before guidelines can be developed for the prevention and treatment of age-related sarcopenia.
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Lv, Huifang; Dong, Wang; Guo, Kangkang; Jin, Mingxing; Li, Xiaomeng; Li, Cunfa; Zhang, Yanming
2018-06-05
Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious and high-mortality viral disease, causing huge economic losses in the swine industry worldwide. CSFV non-structural protein 3 (NS3), a multifunctional protein, plays crucial roles in viral replication. However, how NS3 exactly exerts these functions is currently unknown. Here, we identified tumor necrosis factor receptor-associated factor 5 (TRAF5) as a novel binding partner of the NS3 protein via yeast two-hybrid, co-immunoprecipitation and glutathione S -transferase pull-down assays. Furthermore, we observed that TRAF5 promoted CSFV replication in porcine alveolar macrophages (PAMs). Additionally, CSFV infection or NS3 expression upregulated TRAF5 expression, implying that CSFV may exploit TRAF5 via NS3 for better growth. Moreover, CSFV infection and TRAF5 expression activated p38 mitogen activated protein kinase (MAPK) activity, and inhibition of p38 MAPK activation by the SB203580 inhibitor suppressed CSFV replication. Notably, TRAF5 overexpression did not promote CSFV replication following inhibition of p38 MAPK activation. Our findings reveal that TRAF5 promotes CSFV replication via p38 MAPK activation. This work provides a novel insight into the role of TRAF5 in CSFV replication capacity.
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CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.
Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U
2000-09-12
Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.
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Lv, Gen-fa; Dong, Mao-long; Hu, Da-hai; Zhang, Wan-fu; Wang, Yun-chuan; Tang, Chao-wu; Zhu, Xiong-xiang
2011-01-01
Thermal injury inhibits Akt activation and upregulates p38 mitogen-activated protein kinase, which in turn induces inflammation and increases apoptosis. This study aimed to elucidate the mechanism underlying the cytoprotective role of insulin in severe burns by examining the effects of insulin on inflammation and apoptosis mediated by p38 mitogen-activated protein kinase in burn serum-challenged cardiomyocytes. Neonatal rat cardiomyocytes were exposed to burn serum for 6 hours in the presence or absence of insulin and pretreated with inhibitors to p38 mitogen-activated protein kinase (SB203580) and Akt (LY294002). The authors examined expression of myocardial tumor necrosis factor-alpha, cardiac myofilament proteins caspase-3 and Bcl2, and apoptosis. Burn serum-induced upregulation of tumor necrosis factor was inhibited by both SB203580 and insulin. LY294002 reversed insulin-mediated downregulation of tumor necrosis factor. Both SB203580 and insulin inhibited apoptosis, resulting in fewer pyknotic nuclei and inhibition of caspase-3 activation and Bcl2 downregulation. LY294002 reversed insulin-mediated inhibition of apoptosis. Insulin decreases inflammatory cytokine expression and apoptosis via PI3K/Akt-mediated inhibition of p38 mitogen-activated protein kinase. The cytoprotective role of insulin suggests that it may have a potential role in strategies for treating thermal injuries.
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Jung, Gyung Ah; Shin, Bong Shik; Jang, Yeon Sue; Sohn, Jae Bum; Woo, Seon Rang; Kim, Jung Eun; Choi, Go; Lee, Kyung Mi; Min, Bon Hong; Lee, Kee Ho; Park, Gil Hong
2011-10-31
Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)- p21Cip/WAF1 activation, and suppressed by the mitogenactivated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.
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Kroes, R A; Abravaya, K; Seidenfeld, J; Morimoto, R I
1991-01-01
Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes. Images PMID:2052560
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Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators
NASA Technical Reports Server (NTRS)
Belaguli, N. S.; Sepulveda, J. L.; Nigam, V.; Charron, F.; Nemer, M.; Schwartz, R. J.
2000-01-01
Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.
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Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi
2003-01-01
Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.
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Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients.
Yamamoto, N; Naraparaju, V R; Asbell, S O
1996-06-15
Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices.
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Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B
DOE Office of Scientific and Technical Information (OSTI.GOV)
Reimers, Kerstin; Buchholz, Katja; Werchau, Hermann
2005-01-20
Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cellsmore » revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.« less
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Golas, Avantika; Yeh, Chyi-Huey Josh; Pitakjakpipop, Harit; Siedlecki, Christopher A.; Vogler, Erwin A.
2012-01-01
Activation of blood plasma coagulation in vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. The only plausible explanation consistent with current understanding of coagulation-cascade biochemistry is that procoagulant stimulus arising from the activation complex of the intrinsic pathway is dependent on activator surface area. And yet, it is herein shown that activation of the blood zymogen factor XII (Hageman factor, FXII) dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a “mechanochemical” reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results of this study strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway. PMID:23117212
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Shi, Junwei; Zhang, Huan; Fang, Liurong
Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferasemore » by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.« less
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Andersson, Helena M.; Arantes, Márcia J.; Crawley, James T. B.; Luken, Brenda M.; Tran, Sinh; Dahlbäck, Björn; Rezende, Suely M.
2010-01-01
Protein S has an established role in the protein C anticoagulant pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (APC). Despite its physiological role and clinical importance, the molecular basis of its action is not fully understood. To clarify the mechanism of the protein S interaction with APC, we have constructed and expressed a library of composite or point variants of human protein S, with residue substitutions introduced into the Gla, thrombin-sensitive region (TSR), epidermal growth factor 1 (EGF1), and EGF2 domains. Cofactor activity for APC was evaluated by calibrated automated thrombography (CAT) using protein S–deficient plasma. Of 27 variants tested initially, only one, protein S D95A (within the EGF1 domain), was largely devoid of functional APC cofactor activity. Protein S D95A was, however, γ-carboxylated and bound phospholipids with an apparent dissociation constant (Kdapp) similar to that of wild-type (WT) protein S. In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to have greatly reduced ability to enhance APC-induced cleavage of FVa Arg306. It is concluded that residue Asp95 within EGF1 is critical for APC cofactor function of protein S and could define a principal functional interaction site for APC. PMID:20308596
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Wang, Xuping; Zheng, Rongzong; Huang, Xiaowen; Mao, Zhujun; Wang, Nani; Li, Hongyu; Wen, Chengping; Shou, Dan
2018-03-25
Chronic osteomyelitis is primarily caused by infection with Staphylococcus aureus (S. aureus). Antibiotics are commonly administered; however, it is a challenge to promote bone healing. The aim of this study was to investigate the in vitro effects of alkaloids from the herbal remedy Sophora flavescens (ASF) on rat calvarial osteoblasts (ROBs) infected with S. aureus and healthy osteoclasts. Cell proliferation and alkaline phosphatase, interleukin-6, and tumour necrosis factor-α activity was measured in infected ROBs; tartrate-resistant acid phosphatase was evaluated in osteoclasts via enzyme-linked immunosorbent assay. The mRNA and protein expression levels of bone morphogenetic protein 2, runt-related transcription factor 2, osteoprotegerin, and receptor activator of nuclear factor kappa-B ligand were assessed in infected ROBs through reverse transcription-polymerase chain reaction and western blotting analysis, respectively. Results indicated that ASF increased the viability of uninfected ROBs and infected ROBs treated with vancomycin via regulation of bone morphogenetic protein 2, runt-related transcription factor, osteoprotegerin, and receptor activator of nuclear factor kappa-B ligand mRNA and protein expression levels. In addition, the secretion of the inflammatory factor tumour necrosis factor-α was decreased and alkaline phosphatase activity was increased, inhibiting the viability of osteoclasts and tartrate-resistant acid phosphatase activity. Therefore, the herbal remedy ASF has potential as a new treatment for chronic osteomyelitis. Copyright © 2018 John Wiley & Sons, Ltd.
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Lin, Changsheng; Ear, Jason; Midde, Krishna; Lopez-Sanchez, Inmaculada; Aznar, Nicolas; Garcia-Marcos, Mikel; Kufareva, Irina; Abagyan, Ruben; Ghosh, Pradipta
2014-01-01
A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs. PMID:25187647
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Pirat, Bahar; Muderrisoglu, Haldun; Unal, Muge Tecder; Ozdemir, Handan; Yildirir, Aylin; Yucel, Muammer; Turkoglu, Suna
2007-02-01
Myocardial apoptosis is recognized as a major mechanism of cell death during ischemia-reperfusion. In this study, we assessed the hypothesis that activated protein C may have a cardioprotective effect via preventing apoptosis in a rat model of myocardial ischemia-reperfusion. Thirty male Sprague-Dawley rats were anesthetized, instrumented for hemodynamic measurements and ventilated mechanically. Twenty rats were subjected to 20 min of left anterior descending coronary artery occlusion and 2 h of reperfusion. They were randomly assigned to receive intravenous Ringer lactate (vehicle) or activated protein C (2 mg/kg/h) 10 min after occlusion and during reperfusion. The other 10 rats were sham-operated. At the end of the reperfusion period, serum samples were obtained for evaluation of creatine kinase, C-reactive protein and tumor necrosis factor-alpha. Apoptosis was measured quantitatively by the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling method. Serum creatine kinase, C-reactive protein and tumor necrosis factor-alpha values and percentage of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling- positive myocyte nuclei demonstrated negligible myocardial injury in sham-operated controls. During reperfusion, mean arterial pressures were significantly higher in activated protein C-treated rats than in the control group (68.2+/-10.3 vs. 55.4+/-11.6 mmHg, P=0.01). Number of apoptotic cells was significantly reduced from 47.7 to 24.8% with activated protein C administration (P=0.008). No difference was seen between activated protein C-treated and untreated animals with respect to creatine kinase, C-reactive protein and tumor necrosis factor-alpha levels. Treatment with activated protein C significantly improved hemodynamics after ischemia-reperfusion and reduced ischemia-reperfusion-induced myocardial apoptosis in rats.
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McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A
1992-01-01
The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092
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Effect of platelet-derived β-thromboglobulins on coagulation.
Egan, Karl; van Geffen, Johanna P; Ma, Hui; Kevane, Barry; Lennon, Aine; Allen, Seamus; Neary, Elaine; Parsons, Martin; Maguire, Patricia; Wynne, Kieran; O' Kennedy, Richard; Heemskerk, Johan W M; Áinle, Fionnuala Ní
2017-06-01
β-thromboglobulins are derived from the cleavage of the CXC chemokine platelet basic protein and are released in high concentrations by activated platelets. Platelet-derived β-thromboglobulins (βTG) share 70% homology with platelet factor 4 (PF4), another CXC chemokine released by activated platelets. PF4 modulates coagulation by inhibiting heparin-antithrombin interactions, promoting protein C activation, and attenuating the activity of activated protein C. In contrast, the effect of βTG on coagulation is unknown. Clotting times, thrombin generation, chromogenic clotting factor assays, and surface plasmon resonance (SPR) were used to assess the effect of purified βTG on coagulation. In normal pooled plasma, βTG shortened the lagtime and time to peak thrombin generation of tissue factor (TF)-dependent and TF-independent thrombin generation. In factor VIII and factor IX-deficient plasmas, βTG induced thrombin generation in the absence of a TF stimulus and in the presence of anti-TF and factor VIIa inhibitory antibodies. The procoagulant effect was not observed when thrombin generation was independent of factor X activation (supplementation of factor X-deficient plasma with factor Xa). Cleavage of a factor Xa-specific chromogenic substrate was observed when βTG was incubated with factor X, suggesting a direct interaction between βTG and factor X. Using SPR, βTG were found to bind to immobilised factor X in a dose dependent manner. βTG modulate coagulation in vitro via an interaction with factor X. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena
2015-01-01
To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins. © 2015 S. Karger AG, Basel.
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van Ooij, C; Snyder, R C; Paeper, B W; Duester, G
1992-01-01
The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver. Images PMID:1620113
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1994-01-01
Cells of monocytic lineage can initiate extravascular fibrin deposition via expression of blood coagulation mediators. This report is about experiments on three mechanisms with the potential to modulate monocyte- initiated coagulation. Monocyte procoagulant activity was examined as a function of lipid cofactor, protein cofactor, and specific inhibitor expression during short-term culture in vitro. Lipid cofactor activity was measured as the initial rate of factor X activation by intrinsic- pathway components, the assembly of which depends on this cofactor. Lipid cofactor activity levels changed by < 30% during 48-h culture. Protein cofactor, i.e., tissue factor (TF) antigen was measured by enzyme immunoassay. It increased from 461 pg/ml to a maximum value of 3,550 pg/ml at 24 h and remained at 70% of this value. Specific TF activity, measured as factor VII-dependent factor X activation rate, decreased from 54 to 18 nM FXa/min between 24 and 48 h. TF activity did not correlate well with either lipid cofactor or TF protein levels. In contrast, the decrease in TF activity coincided in time with maximal expression of tissue factor pathway inhibitor (TFPI) mRNA, which was determined using reverse transcriptase polymerase chain reaction (RT- PCR), and with maximal TFPI protein levels measured by immunoassay. The number of mRNA copies coding for TFPI and TF in freshly isolated blood monocytes were 46 and 20 copies/cells, respectively. These values increased to 220 and 63 copies/cell during short-term cell culture in the presence of endotoxin. Results demonstrate concomitant expression by monocytes of genes coding for both the essential protein cofactor and the specific inhibitor of the extrinsic coagulation pathway. Together with functional and antigenic analyses, they also imply that the initiation of blood clotting by extravascular monocyte/macrophages can be modulated locally by TFPI independently of plasma sources of the inhibitor. PMID:8195712
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Advanced glycation end products and the progressive course of renal disease.
Heidland, A; Sebekova, K; Schinzel, R
2001-10-01
In experimental and human diabetic nephropathy (DN), it has been shown that advanced glycation end products (AGEs), in particular, carboxymethyl-lysine and pentosidine, accumulate with malondialdehyde in glomerular lesions in relation to disease severity and in the presence of an upregulated receptor for AGE (RAGE) in podocytes. Toxic effects of AGEs result from structural and functional alterations in plasma and extracellular matrix (ECM) proteins, in particular, from cross-linking of proteins and interaction of AGEs with their receptors and/or binding proteins. In mesangial and endothelial cells, the AGE-RAGE interaction caused enhanced formation of oxygen radicals with subsequent activation of nuclear factor-kappaB and release of pro-inflammatory cytokines (interleukin-6, tumor necrosis factor-alpha), growth factors (transforming growth factor-beta1 [TGF-beta1], insulin-like growth factor-1), and adhesion molecules (vascular cell adhesion molecule-1, intercellular adhesion molecule-1). In tubular cells, incubation with AGE albumin was followed by stimulation of the mitogen-activating protein (MAP) kinase pathway and its downstream target, the activating protien-1 (AP-1) complex, TGF-beta1 overexpression, enhanced protein kinase C activity, decreased cell proliferation, and impaired protein degradation rate, in part caused by decreased cathepsin activities. The pathogenic relevance of AGEs was further verified by in vivo experiments in euglycemic rats and mice by the parenteral administration of AGE albumin, leading in the glomeruli to TGF-beta1 overproduction, enhanced gene expression of ECM proteins, and morphological lesions similar to those of DN. Evidence for the pathogenic relevance of AGEs in DN also comes from experimental studies in which the formation and/or action of AGEs was modulated by aminoguanidine, OPB-9195, pyridoxamine, soluble RAGEs, serine protease trypsin, and antioxidants, resulting in improved cell and/or renal function.
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Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Kubo, Shinichi; Hori, Hitoshi
2004-01-01
Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.
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CIKS, a connection to IκB kinase and stress-activated protein kinase
Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich
2000-01-01
Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033
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Parrish, William; Eilers, Markus; Ying, Weiwen; Konopka, James B
2002-01-01
The binding of alpha-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the alpha-factor receptor that includes transmembrane domains 1-5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the alpha-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors. PMID:11861550
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Yamamoto, Nobuto; Suyama, Hirofumi; Nakazato, Hiroaki; Yamamoto, Nobuyuki; Koga, Yoshihiko
2008-07-01
Serum vitamin D binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of colorectal cancer patients was lost or reduced because Gc protein is deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Deglycosylated Gc protein cannot be converted to MAF, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage-activating factor (GcMAF) ever discovered, but it produces no side effect in humans. Macrophages treated with GcMAF (100 microg/ml) develop an enormous variation of receptors and are highly tumoricidal to a variety of cancers indiscriminately. Administration of 100 nanogram (ng)/ human maximally activates systemic macrophages that can kill cancerous cells. Since the half-life of the activated macrophages is approximately 6 days, 100 ng GcMAF was administered weekly to eight nonanemic colorectal cancer patients who had previously received tumor-resection but still carried significant amounts of metastatic tumor cells. As GcMAF therapy progressed, the MAF precursor activities of all patients increased and conversely their serum Nagalase activities decreased. Since serum Nagalase is proportional to tumor burden, serum Nagalase activity was used as a prognostic index for time course analysis of GcMAF therapy. After 32-50 weekly administrations of 100 ng GcMAF, all colorectal cancer patients exhibited healthy control levels of the serum Nagalase activity, indicating eradication of metastatic tumor cells. During 7 years after the completion of GcMAF therapy, their serum Nagalase activity did not increase, indicating no recurrence of cancer, which was also supported by the annual CT scans of these patients.
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Inhibition of Interferon Regulatory Factor 3 Activation by Paramyxovirus V Protein
Irie, Takashi; Kiyotani, Katsuhiro; Igarashi, Tomoki; Yoshida, Asuka
2012-01-01
The V protein of Sendai virus (SeV) suppresses innate immunity, resulting in enhancement of viral growth in mouse lungs and viral pathogenicity. The innate immunity restricted by the V protein is induced through activation of interferon regulatory factor 3 (IRF3). The V protein has been shown to interact with melanoma differentiation-associated gene 5 (MDA5) and to inhibit beta interferon production. In the present study, we infected MDA5-knockout mice with V-deficient SeV and found that MDA5 was largely unrelated to the innate immunity that the V protein suppresses in vivo. We therefore investigated the target of the SeV V protein. We previously reported interaction of the V protein with IRF3. Here we extended the observation and showed that the V protein appeared to inhibit translocation of IRF3 into the nucleus. We also found that the V protein inhibited IRF3 activation when induced by a constitutive active form of IRF3. The V proteins of measles virus and Newcastle disease virus inhibited IRF3 transcriptional activation, as did the V protein of SeV, while the V proteins of mumps virus and Nipah virus did not, and inhibition by these proteins correlated with interaction of each V protein with IRF3. These results indicate that IRF3 is important as an alternative target of paramyxovirus V proteins. PMID:22532687
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Genetics Home Reference: anhidrotic ectodermal dysplasia with immune deficiency
... The proteins produced from these two genes regulate nuclear factor-kappa-B. Nuclear factor-kappa-B is a group of related ... proteins with impaired function, which reduces activation of nuclear factor-kappa-B. These changes disrupt certain signaling ...
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu
2015-03-01
Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interruptsmore » the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.« less
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Datta, Moumita; Bhattacharyya, Nitai P.
2011-01-01
Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. PMID:21832040
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Datta, Moumita; Bhattacharyya, Nitai P
2011-09-30
Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease.
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Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF1
Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki
2008-01-01
Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461
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Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF.
Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki
2008-07-01
Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years.
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Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB
Fiume, Giuseppe; Rossi, Annalisa; de Laurentiis, Annamaria; Falcone, Cristina; Pisano, Antonio; Vecchio, Eleonora; Pontoriero, Marilena; Scala, Iris; Scialdone, Annarita; Masci, Francesca Fasanella; Mimmi, Selena; Palmieri, Camillo; Scala, Giuseppe; Quinto, Ileana
2013-01-01
Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control. PMID:23776612
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Goldman, D; Sapru, M K; Stewart, S; Plotkin, J; Libermann, T A; Wasylyk, B; Guan, K
1998-10-15
An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.
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Emerging technologies for enabling proangiogenic therapy
NASA Astrophysics Data System (ADS)
Sinha Roy, Rituparna; Roy, Bhaskar; Sengupta, Shiladitya
2011-12-01
Ischemic disease causes a large number of deaths and significant clinical problems worldwide. Therapeutic angiogenesis, strengthened by advances in growth-factor-based therapies, is a promising solution to ischemic pathologies. Major challenges in therapeutic angiogenesis are the lack of stability of native angiogenic proteins and also providing sustained delivery of biologically active proteins at the ischemic sites. This paper will discuss various protein engineering strategies to develop stabilized proangiogenic proteins and several biomaterial technologies used to amplify the angiogenic outcome by delivering biologically active growth factors in a sustained manner.
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N-3 polyunsaturated fatty acid regulation of hepatic gene transcription
Jump, Donald B.
2009-01-01
Purpose of review The liver plays a central role in whole body lipid metabolism and adapts rapidly to changes in dietary fat composition. This adaption involves changes in the expression of genes involved in glycolysis, de-novo lipogenesis, fatty acid elongation, desaturation and oxidation. This review brings together metabolic and molecular studies that help explain n-3 (omega-3) polyunsaturated fatty acid regulation of hepatic gene transcription. Recent findings Dietary n-3 polyunsaturated fatty acid regulates hepatic gene expression by targeting three major transcriptional regulatory networks: peroxisome proliferator-activated receptor α, sterol regulatory element binding protein-1 and the carbohydrate regulatory element binding protein/Max-like factor X heterodimer. 22 : 6,n-3, the most prominent n-3 polyunsaturated fatty acid in tissues, is a weak activator of peroxisome proliferator-activated receptor α. Hepatic metabolism of 22 : 6,n-3, however, generates 20 : 5,n-3, a strong peroxisome proliferator-activated receptor α activator. In contrast to peroxisome proliferator-activated receptor α, 22 : 6,n-3 is the most potent fatty acid regulator of hepatic sterol regulatory element binding protein-1. 22 : 6,n-3 suppresses sterol regulatory element binding protein-1 gene expression while enhancing degradation of nuclear sterol regulatory element binding protein-1 through 26S proteasome and Erk1/2-dependent mechanisms. Both n-3 and n-6 polyunsaturated fatty acid suppress carbohydrate regulatory element binding protein and Max-like factor X nuclear abundance and interfere with glucose-regulated hepatic metabolism. Summary These studies have revealed unique mechanisms by which specific polyunsaturated fatty acids control peroxisome proliferator activated receptor α, sterol regulatory element binding protein-1 and carbohydrate regulatory element binding protein/Max-like factor X function. As such, specific metabolic and signal transduction pathways contribute significantly to the fatty acid regulation of these transcription factors and their corresponding regulatory networks. PMID:18460914
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Taylor, Shannon L; Frias-Staheli, Natalia; García-Sastre, Adolfo; Schmaljohn, Connie S
2009-02-01
Hantaviruses such as Hantaan virus (HTNV) and Andes virus cause two human diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, respectively. For both, disease pathogenesis is thought to be immunologically mediated and there have been numerous reports of patients with elevated levels of proinflammatory and inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), in their sera. Multiple viruses have developed evasion strategies to circumvent the host cell inflammatory process, with one of the most prevalent being the disruption of nuclear factor kappa B (NF-kappaB) activation. We hypothesized that hantaviruses might also moderate host inflammation by interfering with this pathway. We report here that the nucleocapsid (N) protein of HTNV was able to inhibit TNF-alpha-induced activation of NF-kappaB, as measured by a reporter assay, and the activation of endogenous p65, an NF-kappaB subunit. Surprisingly, there was no defect in the degradation of the inhibitor of NF-kappaB (IkappaB) protein, nor was there any alteration in the level of p65 expression in HTNV N-expressing cells. However, immunofluorescence antibody staining demonstrated that cells expressing HTNV N protein and a green fluorescent protein-p65 fusion had limited p65 nuclear translocation. Furthermore, we were able to detect an interaction between HTNV N protein and importin alpha, a nuclear import molecule responsible for shuttling NF-kappaB to the nucleus. Collectively, our data suggest that HTNV N protein can sequester NF-kappaB in the cytoplasm, thus inhibiting NF-kappaB activity. These findings, which were obtained using cells transfected with cDNA representing the HTNV N gene, were confirmed using HTNV-infected cells.
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Factor V activation and inactivation by venom proteases.
Rosing, J; Govers-Riemslag, J W; Yukelson, L; Tans, G
2001-01-01
Blood coagulation factor V is a single-chain glycoprotein with M(r) = 330,000 which plays an important role in the procoagulant and anticoagulant pathways. Thrombin activates factor V into factor Va, a two-chain molecule which is composed of a heavy (M(r) = 105,000) and a light chain (M(r) = 71,000/74,000). Factor Va accelerates factor Xa-catalysed prothrombin activation more than 1,000-fold and under physiological conditions the cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C. Factor V can also be activated by a wide variety of snake venoms (e.g. from Vipera species, Naja naja oxiana, Bothrops atrox) and by proteases present in the bristles of a South American caterpillar (Lonomia achelous). Some venoms, notably of Vipera lebetina turanica and Lonomia achelous, contain proteases that are able to inactivate factor V or factor Va. Venom factor V activators are excellent tools in studying the structure-function relationship of factor V(a) and they are also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway. In this review, the structural and functional properties of animal venom factor V activators and inactivators is described. Copyright 2002 S. Karger AG, Basel
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TALE transcription factors during early development of the vertebrate brain and eye.
Schulte, Dorothea; Frank, Dale
2014-01-01
Our brain's cognitive performance arises from the coordinated activities of billions of nerve cells. Despite a high degree of morphological and functional differences, all neurons of the vertebrate central nervous system (CNS) arise from a common field of multipotent progenitors. Cell fate specification and differentiation are directed by multistep processes that include inductive/external cues, such as the extracellular matrix or growth factors, and cell-intrinsic determinants, such as transcription factors and epigenetic modulators of proteins and DNA. Here we review recent findings implicating TALE-homeodomain proteins in these processes. Although originally identified as HOX-cofactors, TALE proteins also contribute to many physiological processes that do not require HOX-activity. Particular focus is, therefore, given to HOX-dependent and -independent functions of TALE proteins during early vertebrate brain development. Additionally, we provide an overview about known upstream and downstream factors of TALE proteins in the developing vertebrate brain and discuss general concepts of how TALE proteins function to modulate neuronal cell fate specification. Copyright © 2013 Wiley Periodicals, Inc.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Khaiboullina, Svetlana F., E-mail: sv.khaiboullina@gmail.com; Morzunov, Sergey P.; Boichuk, Sergei V.
2013-09-01
Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirusmore » triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.« less
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Jiang, Zheng-Yu; Lu, Meng-Chen; You, Qi-Dong
2016-12-22
The transcription factor Nrf2 is the primary regulator of the cellular defense system, and enhancing Nrf2 activity has potential usages in various diseases, especially chronic age-related and inflammatory diseases. Recently, directly targeting Keap1-Nrf2 protein-protein interaction (PPI) has been an emerging strategy to selectively and effectively activate Nrf2. This Perspective summarizes the progress in the discovery and development of Keap1-Nrf2 PPI inhibitors, including the Keap1-Nrf2 regulatory mechanisms, biochemical techniques for inhibitor identification, and approaches for identifying peptide and small-molecule inhibitors, as well as discusses privileged structures and future directions for further development of Keap1-Nrf2 PPI inhibitors.
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Hwangpo, Tracy Anh; Jordan, J Dedrick; Premsrirut, Prem K; Jayamaran, Gomathi; Licht, Jonathan D; Iyengar, Ravi; Neves, Susana R
2012-04-20
Gα(o/i) interacts directly with GRIN (G protein-regulated inducer of neurite outgrowth). Using the yeast two-hybrid system, we identified Sprouty2 as an interacting partner of GRIN. Gα(o) and Sprouty2 bind to overlapping regions of GRIN, thus competing for GRIN binding. Imaging experiments demonstrated that Gα(o) expression promoted GRIN translocation to the plasma membrane, whereas Sprouty2 expression failed to do so. Given the role of Sprouty2 in the regulation of growth factor-mediated MAPK activation, we examined the contribution of the GRIN-Sprouty2 interaction to CB1 cannabinoid receptor regulation of FGF receptor signaling. In Neuro-2A cells, a system that expresses all of the components endogenously, modulation of GRIN levels led to regulation of MAPK activation. Overexpression of GRIN potentiated FGF activation of MAPK and decreased tyrosine phosphorylation of Sprouty2. Pretreatment with G(o/i)-coupled CB1 receptor agonist attenuated subsequent FGF activation of MAPK. Decreased expression of GRIN both diminished FGF activation of MAPK and blocked CB1R attenuation of MAPK activation. These observations indicate that Gα(o) interacts with GRIN and outcompetes GRIN from bound Sprouty. Free Sprouty then in turn inhibits growth factor signaling. Thus, here we present a novel mechanism of how G(o/i)-coupled receptors can inhibit growth factor signaling to MAPK.
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An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development.
Chae, Eunyoung; Tan, Queenie K-G; Hill, Theresa A; Irish, Vivian F
2008-04-01
Plants flower in response to both environmental and endogenous signals. The Arabidopsis LEAFY (LFY) transcription factor is crucial in integrating these signals, and acts in part by activating the expression of multiple floral homeotic genes. LFY-dependent activation of the homeotic APETALA3 (AP3) gene requires the activity of UNUSUAL FLORAL ORGANS (UFO), an F-box component of an SCF ubiquitin ligase, yet how this regulation is effected has remained unclear. Here, we show that UFO physically interacts with LFY both in vitro and in vivo, and this interaction is necessary to recruit UFO to the AP3 promoter. Furthermore, a transcriptional repressor domain fused to UFO reduces endogenous LFY activity in plants, supporting the idea that UFO acts as part of a transcriptional complex at the AP3 promoter. Moreover, chemical or genetic disruption of proteasome activity compromises LFY-dependent AP3 activation, indicating that protein degradation is required to promote LFY activity. These results define an unexpected role for an F-box protein in functioning as a DNA-associated transcriptional co-factor in regulating floral homeotic gene expression. These results suggest a novel mechanism for promoting flower development via protein degradation and concomitant activation of the LFY transcription factor. This mechanism may be widely conserved, as homologs of UFO and LFY have been identified in a wide array of plant species.
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Park, Hyun-Jung; Baen, Ji-Yeon; Lee, Ye-Ji; Choi, Youn-Hee; Kang, Jihee Lee
2012-08-01
The TAM receptor protein tyrosine kinases Tyro3, Axl, and Mer play important roles in macrophage function. We investigated the roles of the TAM receptors in mediating the induction of hepatocyte growth factor (HGF) during the interaction of macrophages with apoptotic cells. Mer-specific neutralizing antibody, small interfering RNA (siRNA), and a recombinant Mer protein (Mer/Fc) inhibited HGF mRNA and protein expression, as well as activation of RhoA, Akt, and specific mitogen-activated protein (MAP) kinases in response to apoptotic cells. Inhibition of Axl or Tyro3 with specific antibodies, siRNA, or Fc-fusion proteins did not prevent apoptotic cell-induced HGF mRNA and protein expression and did not inhibit activation of the postreceptor signaling molecules RhoA and certain MAP kinases, including extracellular signal-regulated protein kinase and c-Jun NH(2)-terminal kinase. However, Axl- and Tyro3-specific blockers did inhibit the activation of Akt and p38 MAP kinase in response to apoptotic cells. In addition, none of the TAM receptors mediated the effects of apoptotic cells on transforming growth factor-β or epidermal growth factor mRNA expression. However, they were involved in the induction of vascular endothelial growth factor mRNA expression. Our data provide evidence that when macrophages interact with apoptotic cells, only Mer of the TAM-family receptors is responsible for mediating transcriptional HGF production through a RhoA-dependent pathway.
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TALE factors poise promoters for activation by Hox proteins.
Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G
2014-01-27
Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription. Copyright © 2014 Elsevier Inc. All rights reserved.
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Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target.
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Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth
2002-09-01
Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.
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Banerjee, A; Emanuel, K; Parafina, J; Bagchi, M
1992-10-01
A water soluble growth inhibitor was isolated from the mammalian ocular iris-ciliary complex. The molecular weight of this protein is 10 kD or lower as determined by ultrafiltration fractionation. The iris-ciliary (IC) complex water soluble protein(s) significantly inhibits synthesis of lower molecular weight proteins of the epithelial cells of the organ cultured mammalian ocular lens. It was also found that this inhibitory effect of IC is mediated via the structural organization of the lens. Monolayer cultures of the lens epithelial cells exposed to IC did not manifest any inhibition of their protein synthesis. Moreover, these tissue cultured lens epithelial (TCLE) cells showed a significant increase in their protein synthetic activities in response to the presence of IC factors in the culture medium. It is postulated that the IC activity is modulated via either the lens capsule, an extracellular matrix, or due to the specific organization of the intact lens. The specific effects of IC on the cytoskeletal organization and synthesis in the organ cultured lens epithelial (OCLE) and TCLE cells were also examined. Both groups, treated with IC factors, manifested significant alterations in their protein synthetic activities and cytoskeletal architecture. The 3H-leucine incorporation experiments showed that alpha-actin and alpha-tubulin synthesis is partially inhibited by IC factors in OCLE cells but vimentin synthesis is not, whereas in TCLE cells all of them showed increased synthesis in response to IC factors. Turnover rates of these proteins in both OCLE and TCLE cells were also computed. The immunofluorescence and microscopic evaluation of OCLE and TCLE cells exposed to IC factors illustrated significant alteration in the cytoarchitecture of the filaments. We demonstrate that an inhibitor(s) molecule of 10 kD or lower size isolated from IC inhibited protein synthesis of OCLE cells and stimulated protein synthesis in TCLE cells. The IC factor also affects the synthesis and organization of cytoskeletal filaments of both the OCLE and TCLE cells.
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Shen, S H; Bastien, L; Posner, B I; Chrétien, P
1991-08-22
The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.
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Src kinase regulation by phosphorylation and dephosphorylation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Roskoski, Robert
2005-05-27
Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shownmore » to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.« less
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Huang, Chien-Hsun; Kuo, Wen-Yu; Jinn, Tsung-Luo
2012-03-01
Copper-zinc superoxide dismutase (CuZnSOD; CSD) is an important antioxidant enzyme for oxidative stress protection. To date, two activation pathways have been identified in many species. One requiring the CCS, Cu chaperone for SOD, to insert Cu and activate CSD (referred to as CCS-dependent pathway), and the other works independently of CCS (referred to as CCS-independent pathway). In our previous study, we suggest an unidentified factor will work with glutathione (GSH) for CSD activation in the absence of the CCS. Here, two models of the CCS-independent mechanism are proposed. The role of the unidentified factor may work as a scaffold protein, which provides a platform for the CSD protein and Cu-GSH to interact, or as a Cu carrier, which itself can bind Cu and interact with CSD proteins. We also suggest that the CSD protein conformation at C-terminal is important in providing a docking site for unidentified factor to access.
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Kuo, Calvin J.; LaMontagne, Kenneth R.; Garcia-Cardeña, Guillermo; Ackley, Brian D.; Kalman, Daniel; Park, Susan; Christofferson, Rolf; Kamihara, Junne; Ding, Yuan-Hua; Lo, Kin-Ming; Gillies, Stephen; Folkman, Judah; Mulligan, Richard C.; Javaherian, Kashi
2001-01-01
Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase–stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis. PMID:11257123
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Schmidt, K H; Gerlach, D; Wollweber, L; Reichardt, W; Mann, K; Ozegowski, J H; Fleischer, B
1995-01-01
M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein. PMID:7591107
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USDA-ARS?s Scientific Manuscript database
Protein elongation factors, EF-Tu and EF-1a, have been implicated in cell response to heat stress. In spring wheat, EF-Tu displays chaperone activity and reduces thermal aggregation of Rubisco activase. Similarly, in mammalian cells, EF-1a displays chaperone-like activity and regulates the expressio...
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Hallenbeck, P C; Meyer, C M; Vignais, P M
1982-01-01
Nitrogenase proteins were isolated from cultures of the photosynthetic bacterium Rhodopseudomonas capsulata grown on a limiting amount of ammonia. Under these conditions, the nitrogenase N2ase A was active in vivo, and nitrogenase activity in vitro was not dependent upon manganese and the activating factor. The nitrogenase proteins were also isolated from nitrogen-limited cultures in which the in vivo nitrogenase activity had been stopped by an ammonia shock. This nitrogenase activity, N2ase R, showed an in vitro requirement for manganese and the activating factor for maximal activity. The Mo-Fe protein (dinitrogenase) was composed of two dissimilar subunits with molecular weights of 55,000 and 59,500; the Fe protein (dinitrogenase reductase), from either type of culture, was composed of a single subunit (molecular weight), 33,500). The metal and acid labile sulfur contents of both nitrogenase proteins were similar to those found for previously isolated nitrogenases. The Fe proteins from both N2ase A and N2ase R contained phosphate and ribose, 2 mol of each per mol of N2ase R Fe protein and about 1 mol of each per mol of N2ase A Fe protein. The greatest difference between the two types of Fe protein was that the N2ase R Fe protein contained about 1 mol per mol of an adenine-like molecule, whereas the N2ase A Fe protein content of this compound was insignificant. These results are compared with various models previously presented for the short-term regulation of nitrogenase activity in the photosynthetic bacteria. PMID:6799495
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Kang, Shin-Ae; Na, Hyelin; Kang, Hyun-Jin; Kim, Sung-Hye; Lee, Min-Ho; Lee, Mi-Ock
2010-09-15
Although the roles of Nur77, an orphan member of the nuclear hormone receptor superfamily, in the control of cellular proliferation, apoptosis, inflammation, and glucose metabolism, are well recognized, the molecular mechanism regulating the activity and expression of Nur77 is not fully understood. Acetylation of transcription factors has emerged recently as a major post-translational modification that regulates protein stability and transcriptional activity. Here, we examined whether Nur77 is acetylated, and we characterized potential associated factors. First, Nur77 was found to be an acetylated protein when examined by immunoprecipitation and western blotting using acetyl protein-specific antibodies. Second, expression of p300, which possesses histone acetyltransferase activity, enhanced the acetylation and protein stability of Nur77. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, also increased Nur77 acetylation. Among the several types of HDACs, HDAC1 was found as the major enzyme affecting protein level of Nur77. HDAC1 decreased the acetylation level, protein level, and transcriptional activity of Nur77. Interestingly, overexpression of Nur77 induced expression of both p300 and HDAC1. Finally, the expression of Nur77 increased along with that of p300, but decreased with induction of HDAC1 after treatment with epithelial growth factor, nerve growth factor, or 6-mercaptopurine, suggesting that the self-control of the acetylation status contributes to the transient induction of Nur77 protein. Taken together, these results demonstrate that acetylation of Nur77 is modulated by p300 and HDAC1, and suggest that acetylation is an important post-translational modification for the rapid turnover of Nur77 protein. Copyright 2010 Elsevier Inc. All rights reserved.
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Wang, Jun; Lee, Seungsoo; Teh, Charis En-Yi; Bunting, Karen; Ma, Lina; Shannon, M Frances
2009-03-01
Activation of T cells leads to the induction of many cytokine genes that are required for appropriate immune responses, including IL-2, a key cytokine for T cell proliferation and homeostasis. The activating transcription factors such as nuclear factor of activated T cells, nuclear factor kappaB/Rel and activated protein-1 family members that regulate inducible IL-2 gene expression have been well documented. However, negative regulation of the IL-2 gene is less studied. Here we examine the role of zinc finger E-box-binding protein (ZEB) 1, a homeodomain/Zn finger transcription factor, as a repressor of IL-2 gene transcription. We show here that ZEB1 is expressed in non-stimulated and stimulated T cells and using chromatin immunoprecipitation assays we show that ZEB1 binds to the IL-2 promoter. Over-expression of ZEB1 can repress IL-2 promoter activity, as well as endogenous IL-2 mRNA production in EL-4 T cells, and this repression is dependent on the ZEB-binding site at -100. ZEB1 cooperates with the co-repressor C-terminal-binding protein (CtBP) 2 and with histone deacetylase 1 to repress the IL-2 promoter and this cooperation depends on the ZEB-binding site in the promoter as well as the Pro-X-Asp-Leu-Ser protein-protein interaction domain in CtBP2. Thus, ZEB1 may function to recruit a repressor complex to the IL-2 promoter.
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Yang, Liu; Sha, Haibo; Davisson, Robin L.; Qi, Ling
2013-01-01
Activation of the unfolded protein response (UPR) is associated with the disruption of endoplasmic reticulum (ER) homeostasis and has been implicated in the pathogenesis of many human metabolic diseases, including obesity and type 2 diabetes. However, the nature of the signals activating UPR under these conditions remains largely unknown. Using a method that we recently optimized to directly measure UPR sensor activation, we screened the effect of various metabolic drugs on UPR activation and show that the anti-diabetic drug phenformin activates UPR sensors IRE1α and pancreatic endoplasmic reticulum kinase (PERK) in both an ER-dependent and ER-independent manner. Mechanistically, AMP-activated protein kinase (AMPK) activation is required but not sufficient to initiate phenformin-mediated IRE1α and PERK activation, suggesting the involvement of additional factor(s). Interestingly, activation of the IRE1α (but not PERK) pathway is partially responsible for the cytotoxic effect of phenformin. Together, our data show the existence of a non-canonical UPR whose activation requires the cytosolic kinase AMPK, adding another layer of complexity to UPR activation upon metabolic stress. PMID:23548904
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Li, Xiaodong; Hoeppner, Luke H.; Jensen, Eric D.; Gopalakrishnan, Rajaram; Westendorf, Jennifer J.
2013-01-01
Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells. PMID:19585539
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Alvarenga, Patricia H.; Xu, Xueqing; Oliveira, Fabiano; Chagas, Andrezza C.; Nascimento, Clarissa R.; Francischetti, Ivo M.B.; Juliano, Maria A.; Juliano, Luiz; Scharfstein, Julio; Valenzuela, Jesus G.; Ribeiro, José M.C.; Andersen, John F.
2014-01-01
Objective Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway. Approach and Results Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface. Conclusions The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism. PMID:24092749
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van Weering, David H. J.; de Rooij, Johan; Marte, Barbara; Downward, Julian; Bos, Johannes L.; Burgering, Boudewijn M. T.
1998-01-01
Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events. PMID:9528752
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ERIC Educational Resources Information Center
Gelinas, Jennifer N.; Banko, Jessica L.; Peters, Melinda M.; Klann, Eric; Weeber, Edwin J.; Nguyen, Peter V.
2008-01-01
cAMP is a critical second messenger implicated in synaptic plasticity and memory in the mammalian brain. Substantial evidence links increases in intracellular cAMP to activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream effectors (transcription factors, receptors, protein kinases) necessary for long-term…
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Tat-APE1/ref-1 protein inhibits TNF-{alpha}-induced endothelial cell activation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung
2008-03-28
Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-{alpha}-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1 h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-{alpha}-induced monocyte adhesion and vascular cell adhesion molecule-1 expressionmore » in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.« less
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Transcriptional switches in the control of macronutrient metabolism.
Wise, Alan
2008-06-01
This review shows how some transcription factors respond to alterations in macronutrients. Carbohydrates induce enzymes for their metabolism and fatty acid synthesis. Fatty acids reduce carbohydrate processing, induce enzymes for their metabolism, and increase both gluconeogenesis and storage of fat. Fat stores help control carbohydrate uptake by other cells. The following main transcription factors are discussed: carbohydrate response element-binding protein; sterol regulatory element-binding protein-1c, cyclic AMP response element-binding protein, peroxisome proliferator-activated receptor-alpha, and peroxisome proliferator-activated receptor-gamma.
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Wang, Michael; Álvarez-Román, María Teresa; Chowdary, Pratima; Quon, Doris V; Schafer, Kim
2016-10-01
The World Federation of Hemophilia and the National Hemophilia Foundation encourage people with haemophilia (PWH) to participate in routine physical activity. The benefits of physical activity for PWH include improvements in joint, bone, and muscle health. Accordingly, a number of studies suggest that levels of physical activity among PWH are similar to those of their healthy peers, especially among individuals who began prophylaxis at an early age (≤3 years). Importantly, several studies found either no increased risk or only a transient increase in risk of bleeding with more intensive physical activity compared with less intensive physical activity. Data on optimal prophylaxis regimens for PWH who participate in physical/sporting activities; however, remain sparse. Long-acting recombinant factor VIII Fc fusion protein (rFVIIIFc) and recombinant factor IX Fc fusion protein (rFIXFc) demonstrated efficacy for the prevention and treatment of bleeding episodes in Phase 3 clinical trials of participants with haemophilia A and B, respectively, with most individuals able to maintain or increase their physical activities. This manuscript reviews the current literature that describes physical activity in PWH. Additionally, case studies are presented to provide supplemental information to clinicians illustrating the use of rFVIIIFc and rFIXFc in physically active patients with haemophilia A and B, respectively. These case reports demonstrate that it is possible for patients to be physically active and maintain good control of their haemophilia with extended interval prophylactic dosing using rFVIIIFc or rFIXFc.
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Wang, Michael; Álvarez-Román, María Teresa; Chowdary, Pratima; Quon, Doris V.; Schafer, Kim
2016-01-01
The World Federation of Hemophilia and the National Hemophilia Foundation encourage people with haemophilia (PWH) to participate in routine physical activity. The benefits of physical activity for PWH include improvements in joint, bone, and muscle health. Accordingly, a number of studies suggest that levels of physical activity among PWH are similar to those of their healthy peers, especially among individuals who began prophylaxis at an early age (≤3 years). Importantly, several studies found either no increased risk or only a transient increase in risk of bleeding with more intensive physical activity compared with less intensive physical activity. Data on optimal prophylaxis regimens for PWH who participate in physical/sporting activities; however, remain sparse. Long-acting recombinant factor VIII Fc fusion protein (rFVIIIFc) and recombinant factor IX Fc fusion protein (rFIXFc) demonstrated efficacy for the prevention and treatment of bleeding episodes in Phase 3 clinical trials of participants with haemophilia A and B, respectively, with most individuals able to maintain or increase their physical activities. This manuscript reviews the current literature that describes physical activity in PWH. Additionally, case studies are presented to provide supplemental information to clinicians illustrating the use of rFVIIIFc and rFIXFc in physically active patients with haemophilia A and B, respectively. These case reports demonstrate that it is possible for patients to be physically active and maintain good control of their haemophilia with extended interval prophylactic dosing using rFVIIIFc or rFIXFc. PMID:27116081
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Nichols, Daniel Brian; Shisler, Joanna L.
2006-01-01
The pluripotent cytokine tumor necrosis factor alpha (TNF-α) binds to its cognate TNF receptor I (TNF-RI) to stimulate inflammation via activation of the NF-κB transcription factor. To prevent the detrimental effects of TNF-α in keratinocytes infected with the molluscum contagiosum virus (MCV), this poxvirus is expected to produce proteins that block at least one step of the TNF-RI signal transduction pathway. One such product, the MC160 protein, is predicted to interfere with this cellular response because of its homology to other proteins that regulate TNF-RI-mediated signaling. We report here that expression of MC160 molecules did significantly reduce TNF-α-mediated NF-κB activation in 293T cells, as measured by gene reporter and gel mobility shift assays. Since we observed that MC160 decreased other NF-κB activation pathways, namely those activated by receptor-interacting protein, TNF receptor-associated factor 2, NF-κB-inducing kinase, or MyD88, we hypothesized that the MC160 product interfered with I kappa kinase (IKK) activation, an event common to multiple signal transduction pathways. Indeed, MC160 protein expression was associated with a reduction in in vitro IKK kinase activity and IKK subunit phosphorylation. Further, IKK1-IKK2 interactions were not detected in MC160-expressing cells, under conditions demonstrated to induce IKK complex formation, but interactions between the MC160 protein and the major IKK subunits were undetectable. Surprisingly, MC160 expression correlated with a decrease in IKK1, but not IKK2 levels, suggesting a mechanism for MC160 disruption of IKK1-IKK2 interactions. MCV has probably retained its MC160 gene to inhibit NF-κB activation by interfering with signaling via multiple biological mediators. In the context of an MCV infection in vivo, MC160 protein expression may dampen the cellular production of proinflammatory molecules and enhance persistent infections in host keratinocytes. PMID:16378960
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Gautam, Avneesh Kumar; Panse, Yogesh; Ghosh, Payel; Reza, Malik Johid; Mullick, Jayati; Sahu, Arvind
2015-01-01
The complement system has evolved to annul pathogens, but its improper regulation is linked with diseases. Efficient regulation of the system is primarily provided by a family of proteins termed regulators of complement activation (RCA). The knowledge of precise structural determinants of RCA proteins critical for imparting the regulatory activities and the molecular events underlying the regulatory processes, nonetheless, is still limited. Here, we have dissected the structural requirements of RCA proteins that are crucial for one of their two regulatory activities, the cofactor activity (CFA), by using the Kaposi’s sarcoma-associated herpesvirus RCA homolog Kaposica as a model protein. We have scanned the entire Kaposica molecule by sequential mutagenesis using swapping and site-directed mutagenesis, which identified residues critical for its interaction with C3b and factor I. Mapping of these residues onto the modeled structure of C3b–Kaposica–factor I complex supported the mutagenesis data. Furthermore, the model suggested that the C3b-interacting residues bridge the CUB (complement C1r-C1s, Uegf, Bmp1) and MG2 (macroglobulin-2) domains of C3b. Thus, it seems that stabilization of the CUB domain with respect to the core of the C3b molecule is central for its CFA. Identification of CFA-critical regions in Kaposica guided experiments in which the equivalent regions of membrane cofactor protein were swapped into decay-accelerating factor. This strategy allowed CFA to be introduced into decay-accelerating factor, suggesting that viral and human regulators use a common mechanism for CFA. PMID:26420870
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Filardo, Edward J
2002-02-01
The biological and biochemical effects of estrogen have been ascribed to its known receptors, which function as ligand-inducible transcription factors. However, estrogen also triggers rapid activation of classical second messengers (cAMP, calcium, and inositol triphosphate) and stimulation of intracellular signaling cascades mitogen-activated protein kinase (MAP K), PI3K and eNOS. These latter events are commonly activated by membrane receptors that either possess intrinsic tyrosine kinase activity or couple to heterotrimeric G-proteins. We have shown that estrogen transactivates the epidermal growth factor receptor (EGFR) to MAP K signaling axis via the G-protein-coupled receptor (GPCR), GPR30, through the release of surface-bound proHB-EGF from estrogen receptor (ER)-negative human breast cancer cells [Molecular Endocrinology 14 (2000) 1649]. This finding is consistent with a growing body of evidence suggesting that transactivation of EGFRs by GPCRs is a recurrent theme in cell signaling. GPCR-mediated transactivation of EGFRs by estrogen provides a previously unappreciated mechanism of cross-talk between estrogen and serum growth factors, and explains prior data reporting the EGF-like effects of estrogen. This novel mechanism by which estrogen activates growth factor-dependent signaling and its implications for breast cancer biology are discussed further in this review.
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Functional domains of the human splicing factor ASF/SF2.
Zuo, P; Manley, J L
1993-01-01
The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5' splice sites in a concentration-dependent manner. ASF/SF2 is the prototype of a family of proteins typified by the presence of one or two RNP-type RNA binding domains (RBDs) and a region highly enriched in repeating arginine-serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regions essential for one, or both, of its two principal activities, and provides insights into how this type of protein functions in splicing. Two isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, thereby functioning as splicing repressors. Some, but not all, mutations in the RS region prevent ASF/SF2 from functioning as an essential splicing factor. However, the entire RS region can be deleted without reducing splice site switching activity, indicating that it is not absolutely required for interaction with other splicing factors. Experiments with deletion and substitution mutants reveal that the protein contains two related, but highly diverged, RBDs, and that both are essential for activity. Each RBD by itself retains the ability to bind RNA, although optimal binding requires both domains. Images PMID:8223481
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Faucheux, M; Roignant, J-Y; Netter, S; Charollais, J; Antoniewski, C; Théodore, L
2003-02-01
Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.
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Faucheux, M.; Roignant, J.-Y.; Netter, S.; Charollais, J.; Antoniewski, C.; Théodore, L.
2003-01-01
Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes. PMID:12556479
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Regulating the ethylene response of a plant by modulation of F-box proteins
Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA
2014-01-07
The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.
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LPS-inducible factor(s) from activated macrophages mediates cytolysis of Naegleria fowleri amoebae
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cleary, S.F.; Marciano-Cabral, F.
1986-03-01
Soluble cytolytic factors of macrophage origin have previously been described with respect to their tumoricidal activity. The purpose of this study was to investigate the mechanism and possible factor(s) responsible for cytolysis of the amoeba Naegleria fowleri by activated peritoneal macrophages from B6C3F1 mice. Macrophages or conditioned medium (CM) from macrophage cultures were incubated with /sup 3/H-Uridine labeled amoebae. Percent specific release of label served as an index of cytolysis. Bacille Calmette-Guerin (BCG) and Corynebacterium parvum macrophages demonstrated significant cytolysis of amoebae at 24 h with an effector to target ratio of 10:1. Treatment of macrophages with inhibitors of RNAmore » or protein synthesis blocked amoebicidal activity. Interposition of a 1 ..mu..m pore membrane between macrophages and amoebae inhibited killing. Inhibition in the presence of the membrane was overcome by stimulating the macrophages with LPS. CM from SPS-stimulated, but not unstimulated, cultures of activated macrophages was cytotoxic for amoebae. The activity was heat sensitive and was recovered from ammonium sulfate precipitation of the CM. Results indicate that amoebicidal activity is mediated by a protein(s) of macrophage origin induced by target cell contact or stimulation with LPS.« less
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Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant
Abstract
HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...
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Ishii, N; Yamamoto, M; Lahm, H W; Iizumi, S; Yoshihara, F; Nakayama, H; Arisawa, M; Aoki, Y
1997-02-01
Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.
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Ohmichi, M; Decker, S J; Saltiel, A R
1992-10-01
Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.
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Protein S is inducible by interleukin 4 in T cells and inhibits lymphoid cell procoagulant activity
Smiley, Stephen T.; Boyer, Sarah N.; Heeb, Mary J.; Griffin, John H.; Grusby, Michael J.
1997-01-01
Extravascular procoagulant activity often accompanies cell-mediated immune responses and systemic administration of pharmacologic anticoagulants prevents cell-mediated delayed-type hypersensitivity reactions. These observations suggest a direct association between coagulation and cell-mediated immunity. The cytokine interleukin (IL)-4 potently suppresses cell-mediated immune responses, but its mechanism of action remains to be determined. Herein we demonstrate that the physiologic anticoagulant protein S is IL-4-inducible in primary T cells. Although protein S was known to inhibit the classic factor Va-dependent prothrombinase assembled by endothelial cells and platelets, we found that protein S also inhibits the factor Va-independent prothrombinase assembled by lymphoid cells. Thus, protein S-mediated down-regulation of lymphoid cell procoagulant activity may be one mechanism by which IL-4 antagonizes cell-mediated immunity. PMID:9326636
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Contact activation: a revision.
Schmaier, A H
1997-07-01
In conclusion, a revised view of the contact system has been presented. This system has little to do with the initiation of hemostasis. Like lupus anticoagulants, deficiencies of contact proteins give prolonged APTTs but may be risk factors for thrombosis. BK from kininogens is a potent modulator of vascular biology inducing vasodilation, tissue plasminogen activator release, and prostacyclin liberation. Kininogens, themselves, are selective inhibitors of alpha-thrombin-induced platelet activation preventing alpha-thrombin from cleaving the cloned thrombin receptor after arginine41. Kininogens' alpha-thrombin inhibitory activity exists in intact kininogens, BK, and all of BK's breakdown products. HK also is the pivotal protein for contact protein assembly on endothelium. It is the receptor for prekallikrein which when bound to HK becomes activated to kallikrein by an endothelial cell enzyme system independent of activated forms of plasma factor XII. Prekallikrein activation on endothelial cells results in kinetically favorable single chain urokinase and plasminogen activation. Thus the "physiologic, negatively charged surface" for contact system activation is really the assembly of these proteins on cell membranes and activation by membrane-associated enzymes.
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Anticoagulatory and antiinflammatory effects of astaxanthin in diabetic rats.
Chan, Kung-Chi; Pen, Pei-Jain; Yin, Mei-Chin
2012-02-01
Astaxanthin at 0.01 or 0.05% of the diet was supplied to diabetic rats for 12 wk. Astaxanthin intake significantly increased its deposit in plasma, and retained glutathione content, reduced the production of reactive oxygen species, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 in blood and kidney of diabetic rats (P < 0.05). Astaxanthin treatments also significantly decreased plasma levels of C-reactive protein and von Willebrand factor in diabetic rats (P < 0.05). Astaxanthin intake at 0.05% significantly diminished plasminogen activator inhibitor-1 and factor VII activities, enhanced antithrombin-III and protein C activities in circulation (P < 0.05). These results support that astaxanthin could attenuate diabetes associated coagulatory, oxidative, and inflammatory stress. © 2012 Institute of Food Technologists®
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Krivoruchko, Anastasia; Storey, Kenneth B
2013-02-01
Red-eared slider turtles, Trachemys scripta elegans, can survive for several weeks without oxygen when submerged in cold water. We hypothesized that anaerobiosis is aided by adaptive up-regulation of the unfolded protein response (UPR), a stress-responsive pathway that is activated by accumulation of unfolded proteins in the endoplasmic reticulum (ER) and functions to restore ER homeostasis. RT-PCR, western immunoblotting and DNA-binding assays were used to quantify the responses and/or activation status of UPR-responsive genes and proteins in turtle tissues after animal exposure to 5 or 20 h of anoxic submergence at 4 °C. The phosphorylation state of protein kinase-like ER kinase (PERK) (a UPR-regulated kinase) and eukaryotic initiation factor 2 (eIF2α) increased by 1.43-2.50 fold in response to anoxia in turtle heart, kidney, and liver. Activation of the PERK-regulated transcription factor, activating transcription factor 4 (ATF4), during anoxia was documented by elevated atf4 transcripts and total ATF4 protein (1.60-2.43 fold), increased nuclear ATF4 content, and increased DNA-binding activity (1.44-2.32 fold). ATF3 and GADD34 (downstream targets of ATF4) also increased by 1.38-3.32 fold in heart and liver under anoxia, and atf3 transcripts were also elevated in heart. Two characteristic chaperones of the UPR, GRP78, and GRP94, also responded positively to anoxia with strong increases in both the transcript and protein levels. The data demonstrate that the UPR is activated in turtle heart, kidney, and liver in response to anoxia, suggesting that this pathway mediates an integrated stress response to protect tissues during oxygen deprivation.
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Hsu, Cheng-Chin; Yang, Hui-Ting; Ho, Jing-Jing; Yin, Mei-Chin; Hsu, Jen-Ying
2016-03-01
The anti-glycative and anti-oxidative effects from Houttuynia cordata leaves aqueous extract (HCAE) in heart and kidney of diabetic mice were examined. HCAE, at 1 or 2 %, was supplied in drinking water for 8 weeks. Plasma glucose and blood urea nitrogen (BUN) levels and creatine phosphokinase (CPK) activity were measured. The production of oxidative and inflammatory factors was determined. Activity and protein expression of associated enzymes or regulators were analyzed. HCAE intake at both doses lowered plasma glucose and BUN levels, and CPK activity and also restored creatinine clearance rate in diabetic mice. HCAE intake, only at 2 %, retained plasma insulin levels (P < 0.05). HCAE reduced reactive oxygen species, protein carbonyl, interleukin-6, tumor necrosis factor-alpha, N (ε) -(carboxymethyl)-lysine, pentosidine and fructose levels, and reserved glutathione content in heart and kidney of diabetic mice (P < 0.05). Diabetes enhanced aldose reductase (AR) activity and protein expression in heart and kidney (P < 0.05). HCAE intake at both doses decreased renal AR activity and protein expression, but only at 2 % lowered cardiac AR activity and protein expression (P < 0.05). Diabetes increased protein expression of RAGE, p47(phox) and gp91(phox), nuclear factor kappa-B (NF-κB) p50, NF-κB p65 and mitogen-activated protein kinase in heart and kidney (P < 0.05). HCAE intake only at 2 % limited RAGE expression, but at 1 and 2 % downregulated p47(phox), NF-κB p65 and p-p38 expression in these organs (P < 0.05). These findings suggest that Houttuynia cordata leaves aqueous extract could ameliorate cardiac and renal injury under diabetic condition.
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Co-transcriptional nuclear actin dynamics
Percipalle, Piergiorgio
2013-01-01
Actin is a key player for nuclear structure and function regulating both chromosome organization and gene activity. In the cell nucleus actin interacts with many different proteins. Among these proteins several studies have identified classical nuclear factors involved in chromatin structure and function, transcription and RNA processing as well as proteins that are normally involved in controlling the actin cytoskeleton. These discoveries have raised the possibility that nuclear actin performs its multi task activities through tight interactions with different sets of proteins. This high degree of promiscuity in the spectrum of protein-to-protein interactions correlates well with the conformational plasticity of actin and the ability to undergo regulated changes in its polymerization states. Several of the factors involved in controlling head-to-tail actin polymerization have been shown to be in the nucleus where they seem to regulate gene activity. By focusing on the multiple tasks performed by actin and actin-binding proteins, possible models of how actin dynamics controls the different phases of the RNA polymerase II transcription cycle are being identified. PMID:23138849
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Yamamoto, N; Naraparaju, V R
1998-06-01
Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice.
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Perez-Ecija, A; Mendoza, F J
2017-11-01
Studies have demonstrated differences in commonly measured haemostatic parameters between donkeys and horses. Whether clotting factors, anticoagulant protein activities and thromboelastography parameters also differ between species is still unknown. To characterise haemostatic parameters in healthy donkeys and to compare these with those in horses. Cross-sectional study. Clotting factors (V, VII, VIII, IX, X, XI and XII), and antithrombin III, Protein C and Protein S activities were measured in 80 healthy Andalusian and crossbred donkeys and 40 healthy Andalusian crossbred horses with assays based on human deficient plasmas. Thromboelastography was performed in 34 donkeys using a coagulation and platelet function analyser. Donkeys had shorter activated partial thromboplastin time (mean ± s.d. 33.4 ± 5.2 s vs. 38.8 ± 4.2 s; P<0.001) and higher Factor VII (1825 ± 206 vs. 1513 ± 174; P<0.001), IX (142 ± 41 vs. 114 ± 28; P<0.05) and XI (59.4 ± 14.0 vs. 27.2 ± 6.3; P<0.001) activities, whereas horses showed higher Factor X (130 ± 32 vs. 145 ± 23; P>0.05) and XII (96 ± 21 vs. 108 ± 15; P<0.001) activities. Antithrombin III (204 ± 26 vs. 174 ± 29; P<0.001), Protein C (33.16 ± 10.0 vs. 7.57 ± 1.70; P<0.001) and Protein S (median [interquartile range]: 7.8 [5.8-9.3] vs. 6.2 [5.2-7.0]; P<0.001) activities were higher in donkeys. Activated clot time (175 [159-189]), time to peak (6.5 [5.8-7.8]) and clot formation rate (26.9 [16.9-36.4]) in donkeys were shorter than reported values in horses. Haemostatic pathways could not be fully evaluated in donkeys because some tests are unavailable. Certain fibrinolytic parameters (plasmin, plasminogen, etc.) have not been characterised in donkeys and this may have affected our results. The haemostatic system in donkeys differs from that in horses and extrapolation of reference values between these species is not appropriate. © 2017 EVJ Ltd.
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Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C
1998-11-01
Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.
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Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.
Yu, Xiaohua; Biedrzycki, Adam H; Khalil, Andrew S; Hess, Dalton; Umhoefer, Jennifer M; Markel, Mark D; Murphy, William L
2017-09-01
Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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2006-09-01
deregulating the function of Par protein complex, we made the unexpected observation that overexpression of Par6 induced growth- factor independent...predisposition factors for human cancer [8] and the human papillomavirus protein E6, targets scribble for degradation[9]. It has also been shown that Par6...vivo and thus is an excellent model to study the important factors in the initiation of the oncogenic process. However, activation of ErbB1 does not
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Shuh, Maureen; Derse, David
2000-01-01
The human T-cell leukemia virus type 1 Tax protein activates the expression of cellular immediate early genes controlled by the serum response element (SRE), which contains both the serum response factor (SRF) binding element (CArG box) and the ternary complex factor (TCF) binding element (Ets box). We show that TCF binding is necessary for Tax activation of the SRE and that Tax directly interacts with TCFs in vitro. In addition, Tax interactions with CREB binding protein (CBP) and p300- and CBP-associated factor were found to be essential for Tax activation of SRF-mediated transcription. PMID:11070040
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Thiel, Gerald; Rössler, Oliver G
2018-06-05
The polyphenol resveratrol is found in many plant and fruits and is a constituent of our diet. Resveratrol has been proposed to have chemopreventive and anti-inflammatory activities. On the cellular level, resveratrol activates stimulus-regulated transcription factors. To identify resveratrol-responsive elements within a natural gene promoter, the molecular pathway leading to c-Fos gene expression by resveratrol was dissected. The c-Fos gene encodes a basic region leucine zipper transcription factor and is a prototype of an immediate-early gene that is regulated by a wide range of signaling molecules. We analyzed chromatin-integrated c-Fos promoter-luciferase reporter genes where transcription factor binding sites were destroyed by point mutations or deletion mutagenesis. The results show that mutation of the binding sites for serum response factor (SRF), activator protein-1 (AP-1) and cAMP response element binding protein (CREB) significantly reduced reporter gene transcription following stimulation of the cells with resveratrol. Inactivation of the binding sites for signal transducer and activator of transcription (STAT) or ternary complex factors did not influence resveratrol-regulated c-Fos promoter activity. Thus, the c-Fos promoter contains three resveratrol-responsive elements, the cAMP response element (CRE), and the binding sites for SRF and AP-1. Moreover, we show that the transcriptional activation potential of the c-Fos protein is increased in resveratrol-stimulated cells, indicating that the biological activity of c-Fos is elevated by resveratrol stimulation. Pharmacological and genetic experiments revealed that the protein kinase ERK1/2 is the signal transducer that connects resveratrol treatment with the c-Fos gene. Copyright © 2018 Elsevier B.V. All rights reserved.
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Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation
Willard, Melinda D; Willard, Francis S; Li, Xiaoyan; Cappell, Steven D; Snider, William D; Siderovski, David P
2007-01-01
Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein α subunits and thus inhibit signaling by many G protein-coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF-mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor-selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation. PMID:17380122
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The role of STATs in transcriptional control and their impact on cellular function.
Bromberg, J; Darnell, J E
2000-05-15
The STAT proteins (Signal Transducers and Activators of Transcription), were identified in the last decade as transcription factors which were critical in mediating virtually all cytokine driven signaling. These proteins are latent in the cytoplasm and become activated through tyrosine phosphorylation which typically occurs through cytokine receptor associated kinases (JAKs) or growth factor receptor tyrosine kinases. Recently a number of non-receptor tyrosine kinases (for example src and abl) have been found to cause STAT phosphorylation. Phosphorylated STATs form homo- or hetero-dimers, enter the nucleus and working coordinately with other transcriptional co-activators or transcription factors lead to increased transcriptional initiation. In normal cells and in animals, ligand dependent activation of the STATs is a transient process, lasting for several minutes to several hours. In contrast, in many cancerous cell lines and tumors, where growth factor dysregulation is frequently at the heart of cellular transformation, the STAT proteins (in particular Stats 1, 3 and 5) are persistently tyrosine phosphorylated or activated. The importance of STAT activation to growth control in experiments using anti-sense molecules or dominant negative STAT protein encoding constructs performed in cell lines or studies in animals lacking specific STATs strongly indicate that STATs play an important role in controlling cell cycle progression and apoptosis. Stat1 plays an important role in growth arrest, in promoting apoptosis and is implicated as a tumor suppressor; while Stats 3 and 5 are involved in promoting cell cycle progression and cellular transformation and preventing apoptosis. Many questions remain including: (1) a better understanding of how the STAT proteins through association with other factors increase transcription initiation; (2) a more complete definition of the sets of genes which are activated by different STATs and (3) how these sets of activated genes differ as a function of cell type. Finally, in the context of many cancers, where STATs are frequently persistently activated, an understanding of the mechanisms leading to their constitutive activation and defining the potential importance of persistent STAT activation in human tumorigenesis remains. Oncogene (2000).
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Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe
2015-01-01
Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles. PMID:26580551
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Li, Fangjun; Li, Maoying; Wang, Ping; Cox, Kevin L; Duan, Liusheng; Dever, Jane K; Shan, Libo; Li, Zhaohu; He, Ping
2017-09-01
Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypium hirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.
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Weyhe, Martin; Eschen-Lippold, Lennart; Pecher, Pascal; Scheel, Dierk; Lee, Justin
2014-01-01
Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.
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Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno
2017-01-01
Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.
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Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R
2016-06-01
Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n TBA exhibit increased ( < 0.05) pAKT protein levels. These data indicate the TBA-mediated increases in protein synthesis likely involve GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBA's effects on fused BSC cultures.
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Morrison, W J; Dhar, A; Shukla, S D
1989-01-01
The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF (100 nM for 5 seconds) stimulated incorporation of 32P into proteins and caused [3H]InsP3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [3H]InsP3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [3H]InsP3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF.
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NASA Astrophysics Data System (ADS)
Yoshida, Tokuyuki; Yoshioka, Yasuo; Morishita, Yuki; Aoyama, Michihiko; Tochigi, Saeko; Hirai, Toshiro; Tanaka, Kota; Nagano, Kazuya; Kamada, Haruhiko; Tsunoda, Shin-ichi; Nabeshi, Hiromi; Yoshikawa, Tomoaki; Higashisaka, Kazuma; Tsutsumi, Yasuo
2015-06-01
Recently, nanomaterial-mediated biological effects have been shown to be governed by the interaction of nanomaterials with some kinds of proteins in biological fluids, and the physical characteristics of the nanomaterials determine the extent and type of their interactions with proteins. Here, we examined the relationships between the surface properties of amorphous silica nanoparticles with diameters of 70 nm (nSP70), their interactions with some proteins in biological fluids, and their toxicity in mice after intravenous administration. The surface modification of nSP70 with amino groups (nSP70-N) prevented acute lethality and abnormal activation of the coagulation cascade found in the nSP70-treated group of mice. Since our previous study showed that coagulation factor XII played a role in the nSP70-mediated abnormal activation of the coagulation cascade, we examined the interaction of nSP70 and nSP70-N with coagulation factor XII. Coagulation factor XII bonded to the surface of nSP70 to a greater extent than that observed for nSP70-N, and consequently more activation of coagulation factor XII was observed for nSP70 than for nSP70-N. Collectively, our results suggest that controlling the interaction of nSP70 with blood coagulation factor XII by modifying the surface properties would help to inhibit the nSP70-mediated abnormal activation of the blood coagulation cascade.
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Macias-Silva, Marina; Li, Wei; Leu, Julia I; Crissey, Mary Ann S; Taub, Rebecca
2002-08-09
Transforming growth factor-beta (TGF-beta) functions as an antiproliferative factor for hepatocytes. However, for unexplained reasons, hepatocytes become resistant to TGF-beta signals and can proliferate despite the presence of TGF-beta during liver regeneration. TGF-beta is up-regulated during liver regeneration, although it is not known whether it is active or latent. TGF-beta activity may be examined by assessing Smad activation, a downstream signaling pathway. Smad pathway activation during liver regeneration induced by partial hepatectomy or CC4 injury was examined by assessing the levels of phospho-Smad2 and Smad2-Smad4 complexes. We found that Smad proteins were slightly activated in quiescent liver, but that their activation was further enhanced in regenerating liver. Interestingly, TGF-beta/Smad pathway inhibitors (SnoN and Ski) were up-regulated during regeneration, and notably, SnoN was induced mainly in hepatocytes. SnoN and Ski are transcriptional repressors that may render some cells resistant to TGF-beta via binding Smad proteins. Complexes between SnoN, Ski, and the activated Smad proteins were detected from 2 to 120 h during the major proliferative phase in regenerating liver. Inhibitory complexes decreased after liver mass restitution (5-15 days), suggesting that persistently activated Smad proteins might participate in returning the liver to a quiescent state. Our data show that active TGF-beta/Smad signals are present during regeneration and suggest that SnoN/Ski induction might explain hepatocyte resistance to TGF-beta during the proliferative phase.
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Amsler, K; Kuwada, S K
1999-01-01
Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.
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Shih, Ying-Chun; Chen, Chao-Ling; Zhang, Yan; Mellor, Rebecca L; Kanter, Evelyn M; Fang, Yun; Wang, Hua-Chi; Hung, Chen-Ting; Nong, Jing-Yi; Chen, Hui-Ju; Lee, Tzu-Han; Tseng, Yi-Shuan; Chen, Chiung-Nien; Wu, Chau-Chung; Lin, Shuei-Liong; Yamada, Kathryn A; Nerbonne, Jeanne M; Yang, Kai-Chien
2018-04-13
Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor β1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor β1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor β1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice ( Txndc5 -/- ) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 ( P <0.001) in Txndc5 -/- versus wild-type mice, respectively. The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against β agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure. © 2018 American Heart Association, Inc.
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Meloche, S; Seuwen, K; Pagès, G; Pouysségur, J
1992-05-01
We have examined the phosphorylation and protein kinase activity of p44 mitogen-activated protein kinase (p44mapk) in growth factor-stimulated hamster fibroblasts using a specific antiserum. The activity of p44mapk was stimulated both by receptor tyrosine kinases and G protein-coupled receptors. Detailed kinetics revealed that alpha-thrombin induces a biphasic activation of p44mapk in CCL39 cells: a rapid phase appearing at 5-10 min was followed by a late and sustained phase still elevated after 4 h. Inactivation of alpha-thrombin with hirudin after 30 sec, which prevented DNA synthesis, did not alter the early p44mapk response but completely abolished the late phase. Pretreatment of the cells with pertussis toxin, which inhibits by more than 95% alpha-thrombin-induced mitogenicity, resulted in the complete loss of late phase activity, while the early peak was partially attenuated. Treatment of CCL39 cells with basic fibroblast growth factor also induced a strong activation of p44mapk. Serotonin, which is not a mitogen by its own, had no effect on late phase p44mapk activity, but synergized with basic fibroblast growth factor to induce late kinase response and DNA synthesis. Both early and late phase activation of p44mapk were accompanied by tyrosine phosphorylation of the enzyme. Together, the results indicate that there is a very close correlation between the ability of a growth factor to induce late and sustained p44mapk activation and its mitogenic potential. Therefore, we propose that sustained p44mapk activation is an obligatory event for growth factor-induced cell cycle progression.
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Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli
Alibolandi, Mona; Mirzahoseini, Hasan
2011-01-01
This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution. PMID:21837279
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Tsapakidis, Konstantinos; Vlachostergios, Panagiotis J; Voutsadakis, Ioannis A; Befani, Christina D; Patrikidou, Anna; Hatzidaki, Eleana; Daliani, Danai D; Moutzouris, George; Liakos, Panagiotis; Papandreou, Christos N
2012-06-01
Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome. © 2012 The Japanese Urological Association.
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Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.
Roskoski, Robert
2005-11-11
Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of interaction with several enzymes and adaptor proteins.
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Hurst, H C; Masson, N; Jones, N C; Lee, K A
1990-12-01
Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.
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Bergamo, Elisa; Diani, Erica; Bertazzoni, Umberto; Romanelli, Maria Grazia
2017-01-01
HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.
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Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.
van der Geer, P; Henkemeyer, M; Jacks, T; Pawson, T
1997-01-01
The Ras guanine nucleotide-binding protein functions as a molecular switch in signalling downstream of protein-tyrosine kinases. Ras is activated by exchange of GDP for GTP and is turned off by hydrolysis of bound GTP to GDP. Ras itself has a low intrinsic GTPase activity that can be stimulated by GTPase-activating proteins (GAPs), including p120-Gap and neurofibromin. These GAPs possess a common catalytic domain but contain distinct regulatory elements that may couple different external signals to control of the Ras pathway. p120-Gap, for example, has two N-terminal SH2 domains that directly recognize phosphotyrosine motifs on activated growth factor receptors and cytoplasmic phosphoproteins. To analyze the role of p120-Gap in Ras regulation in vivo, we have used fibroblasts derived from mouse embryos with a null mutation in the gene for p120-Gap (Gap). Platelet-derived growth factor stimulation of Gap-/- cells led to an abnormally large increase in the level of Ras-GTP and in the duration of mitogen-activated protein (MAP) kinase activation compared with wild-type cells, suggesting that p120-Gap is specifically activated following growth factor stimulation. Induction of DNA synthesis in response to platelet-derived growth factor and morphological transformation by the v-src and EJ-ras oncogenes were not significantly affected by the absence of p120-Gap. However, we found that normal tyrosine phosphorylation of p190-rhoGap, a cytoplasmic protein that associates with the p120-Gap SH2 domains, was dependent on the presence of p120-Gap. Our results suggest that p120-Gap has specific functions in downregulating the Ras/MAP kinase pathway following growth factor stimulation, and in modulating the phosphorylation of p190-rhoGap, but is not required for mitogenic signalling. PMID:9121432
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Igarashi, Yoko; Ohnishi, Kohta; Irie, Kazuhiro; Murakami, Akira
2016-01-01
Zerumbone is a sesquiterpene present in Zinger zerumbet. Many studies have demonstrated its marked anti-inflammatory and anti-carcinogenesis activities. Recently, we showed that zerumbone binds to numerous proteins with scant selectivity and induces the expression of heat shock proteins (HSPs) in hepatocytes. To dampen proteo-toxic stress, organisms have a stress-responsive molecular machinery, known as heat shock response. Heat shock factor 1 (HSF1) plays a key role in this protein quality control system by promoting activation of HSPs. In this study, we investigated whether zerumbone-induced HSF1 activation contributes to its anti-inflammatory functions in stimulated macrophages. Our findings showed that zerumbone increased cellular protein aggregates and promoted nuclear translocation of HSF1 for HSP expression. Interestingly, HSF1 down-regulation attenuated the suppressive effects of zerumbone on mRNA and protein expressions of pro-inflammatory genes, including inducible nitric oxide synthase and interlukin-1β. These results suggest that proteo-stress induced by zerumbone activates HSF1 for exhibiting its anti-inflammatory functions.
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Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation
Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd
2016-01-01
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380
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Rapid kinetic BRET measurements to monitor G protein activation by GPCR and non-GPCR proteins.
Maziarz, Marcin; Garcia-Marcos, Mikel
2017-01-01
Heterotrimeric G proteins are central hubs of signal transduction whose activity is controlled by G protein-coupled receptors (GPCRs) as well as by a complex network of regulatory proteins. Recently, bioluminescence resonance energy transfer (BRET)-based assays have been used to monitor real-time activation of heterotrimeric G proteins in cells. Here we describe the use of a previously established BRET assay to monitor G protein activation upon GPCR stimulation and its adaptation to measure G protein activation by non-GPCR proteins, such as by cytoplasmic guanine nucleotide exchange factors (GEFs) like GIV/Girdin. The BRET assay monitors the release of free Gβγ from Gα-Gβγ heterotrimers as a readout of G protein activation, which is readily observable upon agonist stimulation of GPCRs. To control the signal input for non-GPCR activators, we describe the use of a chemically induced dimerization strategy to promote rapid membrane translocation of proteins containing the Gα-binding and -activating (GBA) motif found in some nonreceptor GEFs. The assay described here allows the kinetic measurement of G protein activation with subsecond temporal resolution and to compare the levels of activation induced by GPCR agonists vs those induced by the membrane recruitment of nonreceptor G protein signaling activators. © 2017 Elsevier Inc. All rights reserved.
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Contact activation of blood-plasma coagulation
NASA Astrophysics Data System (ADS)
Golas, Avantika
Surface engineering of biomaterials with improved hemocompatibility is an imperative, given the widespread global need for cardiovascular devices. Research summarized in this dissertation focuses on contact activation of FXII in buffer and blood plasma frequently referred to as autoactivation. The extant theory of contact activation imparts FXII autoactivation ability to negatively charged, hydrophilic surfaces. According to this theory, contact activation of plasma involves assembly of proteins comprising an "activation complex" on activating surfaces mediated by specific chemical interactions between complex proteins and the surface. This work has made key discoveries that significantly improve our core understanding of contact activation and unravel the existing paradigm of plasma coagulation. It is shown herein that contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution exhibits a parabolic profile when scaled as a function of silanized-glass-particle activator surface energy (measured as advancing water adhesion tension t°a=g° Iv costheta in dyne/cm, where g°Iv is water interfacial tension in dyne/cm and theta is the advancing contact angle). Nearly equal activation is observed at the extremes of activator water-wetting properties --36 < t°a < 72 dyne/cm (O° ≤ theta < 120°), falling sharply through a broad minimum within the 20 < t°a < 40 dyne/cm (55° < theta < 75°). Furthermore, contact activation of FXII in buffer solution produces an ensemble of protein fragments exhibiting either procoagulant properties in plasma (proteolysis of blood factor XI or prekallikrein), amidolytic properties (cleavage of s-2302 chromogen), or the ability to suppress autoactivation through currently unknown biochemistry. The relative proportions of these fragments depend on activator surface chemistry/energy. We have also discovered that contact activation is moderated by adsorption of plasma proteins unrelated to coagulation through an "adsorption-dilution" effect that blocks FXII contact with hydrophobic activator surfaces. The adsorption-dilution effect explains the apparent specificity for hydrophilic activators pursued by earlier investigators. Finally a comparison of FXII autoactivation in buffer, serum, protein cocktail, and plasma solutions is shown herein. Activation of blood plasma coagulation in vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. However, activation of factor XII dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a "mechanochemical" reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway.
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Cell Density Control of Staphylococcal Virulence Mediated by an Octapeptide Pheromone
NASA Astrophysics Data System (ADS)
Ji, Guangyong; Beavis, Ronald C.; Novick, Richard P.
1995-12-01
Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr-encoded signal transduction system for which the octapeptide is the ligand.
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Birse, Kenzie D M; Cole, Amy L; Hirbod, Taha; McKinnon, Lyle; Ball, Terry B; Westmacott, Garrett R; Kimani, Joshua; Plummer, Frank; Cole, Alexander M; Burgener, Adam; Broliden, Kristina
2015-01-01
Cationic proteins found in cervicovaginal secretions (CVS) are known to contribute to the early antiviral immune response against HIV-infection in vitro. We here aimed to define additional antiviral factors that are over-expressed in CVS from female sex workers at high risk of infection. CVS were collected from Kenyan HIV-seronegative (n = 34) and HIV-seropositive (n = 12) female sex workers, and were compared with those from HIV-seronegative low-risk women (n = 12). The highly exposed seronegative (HESN) sex workers were further divided into those with less (n = 22) or more (n = 12) than three years of documented sex work. Cationic protein-depleted CVS were assessed for HIV-neutralizing activity by a PBMC-based HIV-neutralizing assay, and then characterized by proteomics. HIV neutralizing activity was detected in all unprocessed CVS, however only CVS from the female sex worker groups maintained its HIV neutralizing activity after cationic protein-depletion. Differentially abundant proteins were identified in the cationic protein-depleted secretions including 26, 42, and 11 in the HESN>3 yr, HESN<3 yr, and HIV-positive groups, respectively. Gene ontology placed these proteins into functional categories including proteolysis, oxidation-reduction, and epidermal development. The proteins identified in this study include proteins previously associated with the HESN phenotype in other cohorts as well as novel proteins not yet associated with anti-HIV activities. While cationic proteins appear to contribute to the majority of the intrinsic HIV neutralizing activity in the CVS of low-risk women, a broader range of non-cationic proteins were associated with HIV neutralizing activity in HESN and HIV-positive female sex workers. These results indicate that novel protein factors found in CVS of women with high-risk sexual practices may have inherent antiviral activity, or are involved in other aspects of anti-HIV host defense, and warrant further exploration into their mode of action.
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Xu, Weidong; Angelis, Konstantina; Danielpour, David; Haddad, Maher M.; Bischof, Oliver; Campisi, Judith; Stavnezer, Ed; Medrano, Estela E.
2000-01-01
The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type β transforming growth factor (TGF-β) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-β. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-β-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-β-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-β-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-β-mediated growth inhibition in a prostate-derived epithelial cell line. PMID:10811875
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Xu, W; Angelis, K; Danielpour, D; Haddad, M M; Bischof, O; Campisi, J; Stavnezer, E; Medrano, E E
2000-05-23
The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line.
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TAFII-independent activation mediated by human TBP in the presence of the positive cofactor PC4.
Wu, S Y; Kershnar, E; Chiang, C M
1998-01-01
TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs. PMID:9687514
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Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription
DOE Office of Scientific and Technical Information (OSTI.GOV)
Montgomery, K.F.; Tarr, P.I.; Bomsztyk, K.
1991-08-01
Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor {alpha} (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), the authors demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5{prime} flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-{kappa}B and AP-1. Gel mobility shiftmore » assays demonstrate that TNF, IL-1, or LPS induces activation of NF-{kappa}B-like DNA binding activity in HUVEC. Phorbol 12-myristate 13-acetate, a known activator of protein kinase C (PKC), weakly induces NF-{kappa}B-like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-{kappa}B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kB-like proteins but does inhibit ELAM-1 gene transcription. They conclude that PKC-independent activation of NF-{kappa}B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription.« less
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Whisenant, Thomas C; Peralta, Eigen R; Aarreberg, Lauren D; Gao, Nina J; Head, Steven R; Ordoukhanian, Phillip; Williamson, Jamie R; Salomon, Daniel R
2015-01-01
Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
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Aarreberg, Lauren D.; Gao, Nina J.; Head, Steven R.; Ordoukhanian, Phillip; Williamson, Jamie R.; Salomon, Daniel R.
2015-01-01
Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as “central” interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, “peripheral” interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation. PMID:26641092
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Wallqvist, Anders; Memišević, Vesna; Zavaljevski, Nela; Pieper, Rembert; Rajagopala, Seesandra V; Kwon, Keehwan; Yu, Chenggang; Hoover, Timothy A; Reifman, Jaques
2015-12-29
Francisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms. We initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response. Direct interactions with specific host proteins and the ability to influence interactions among host proteins are important components for F. tularensis to avoid host-cell defense mechanisms and successfully establish an infection. Although direct host-pathogen protein-protein binding is only one aspect of Francisella virulence, it is a critical component in directly manipulating and interfering with cellular processes in the host cell.
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PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65
Zhang, Tiejun; Park, Kyeong Ah; Li, Yuwen; Byun, Hee Sun; Jeon, Juhee; Lee, Yoonjung; Hong, Jang Hee; Kim, Jin Man; Huang, Song-Mei; Choi, Seung-Won; Kim, Sun-Hwan; Sohn, Kyung-Cheol; Ro, Hyunju; Lee, Ji Hoon; Lu, Tao; Stark, George R.; Shen, Han-Ming; Liu, Zheng-gang; Park, Jongsun; Hur, Gang Min
2014-01-01
Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that plant homeodomain finger protein 20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In plant homeodomain finger protein 20-overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between plant homeodomain finger protein 20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that plant homeodomain finger protein 20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies plant homeodomain finger protein 20 as a novel regulator of NF-κB activation and suggests that elevated expression of plant homeodomain finger protein 20 may drive constitutive NF-κB activation in some cancers. PMID:23797602
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Structural modification of serum vitamin D3-binding protein and immunosuppression in AIDS patients.
Yamamoto, N; Naraparaju, V R; Srinivasula, S M
1995-11-01
A serum glycoprotein, vitamin D3-binding protein (Gc protein), can be converted by beta-galactosidase of stimulated B lymphocytes and sialidase of T lymphocytes to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is a precursor for MAF. Treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high-titered MAF (GcMAF). When peripheral blood monocytes/macrophages of 46 HIV-infected patients were treated with GcMAF (100 pg/ml), the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of plasma Gc protein was low in 16 (35%) of of these patients. Loss of the MAF precursor activity appeared to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase found in the patient blood stream. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Thus, precursor activity of Gc protein and alpha-N-acetylgalactosaminidase activity in patient blood can serve as diagnostic and prognostic indices.
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Chunhui, Yang; Guohui, Bian; Hong, Yang; Xiaopu, Xiao; Zherong, Bai; Mingyuan, Wang; Xinsheng, Zhang; Juanjuan, Wang; Changqing, Li; Wuping, Li
2013-12-01
Pathogen reduction technology is an important process in the blood safety system, including solvent/detergent treatment, filtration and methylene blue-photochemical technology (MB-PCT). To investigate the quality of MB-PCT-treated plasma, plasma samples from four Chinese blood centers were analyzed over 12 months of storage to determine the recovery of activities and levels of various plasma proteins. Ten plasma units each from the Suzhou, Yancheng, Chongqing and Shandong blood centers were divided into two aliquots. One was subjected to treatment with one of two methylene blue-photochemical technology instruments and the other was used as control. The treated and untreated sample pairs were stored at -30°C. The recovery rates of coagulation factors, inhibitor proteins, total protein, immunoglobulins, and complement proteins were measured at different time points after storage. The mean recovery of most proteins exceeded 80% after MB treatment. The exceptions were factor XI and fibrinogen, of which only 71.3-74% and 69.0-92.3% were retained during storage. The two equipment types differed in terms of residual MB concentration in the plasma samples (0.18 μM and 0.29 μM, respectively). They had similar protein recovery rates at 0.5 month but differed at later time points. The four blood centers differed significantly with regard to factor II, V, VIII and fibrinogen activities. Only the samples from the Shandong blood center met the methylene blue treated fresh frozen plasma requirement. The major factor that influenced the quality of the MB-FFP samples was the time taken between blood collection and storage. Methylene blue treated plasma showed reduced coagulation factor (CF) activity and protein levels. The MB treatment-induced damage to the proteins was acceptable at the four Chinese blood centers, but the quality of the MB-treated plasma in general was not satisfactory. The main factor affecting plasma quality may be the duration of the collection and processing. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Biologically active LIL proteins built with minimal chemical diversity
Heim, Erin N.; Marston, Jez L.; Federman, Ross S.; Edwards, Anne P. B.; Karabadzhak, Alexander G.; Petti, Lisa M.; Engelman, Donald M.; DiMaio, Daniel
2015-01-01
We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context. PMID:26261320
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Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan
2015-05-30
Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.
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Kunin, Margarita; Dmitrieva, Natalia I; Gallazzini, Morgan; Shen, Rong-Fong; Wang, Guanghui; Burg, Maurice B; Ferraris, Joan D
2010-08-11
Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins. We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation. We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity. MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of that factor's transcriptional activity.
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Wilson, Fiona A.; Suryawan, Agus; Orellana, Renán A.; Nguyen, Hanh V.; Jeyapalan, Asumthia S.; Gazzaneo, Maria C.; Davis, Teresa A.
2008-01-01
Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 μg·kg−1·day−1) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P < 0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P < 0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1·eIF4E complex association, and increased active eIF4E·eIF4G complex formation (P < 0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex. PMID:18682537
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Wilson, Fiona A; Suryawan, Agus; Orellana, Renán A; Nguyen, Hanh V; Jeyapalan, Asumthia S; Gazzaneo, Maria C; Davis, Teresa A
2008-10-01
Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.
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Up-regulation of Ciliary Neurotrophic Factor in Astrocytes by Aspirin
Modi, Khushbu K.; Sendtner, Michael; Pahan, Kalipada
2013-01-01
Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor, and the mechanisms by which CNTF expression could be increased in the brain are poorly understood. Acetylsalicylic acid (aspirin) is one of the most widely used analgesics. Interestingly, aspirin increased mRNA and protein expression of CNTF in primary mouse and human astrocytes in a dose- and time-dependent manner. Aspirin induced the activation of protein kinase A (PKA) but not protein kinase C (PKC). H-89, an inhibitor of PKA, abrogated aspirin-induced expression of CNTF. The activation of cAMP-response element-binding protein (CREB), but not NF-κB, by aspirin, the abrogation of aspirin-induced expression of CNTF by siRNA knockdown of CREB, the presence of a consensus cAMP-response element in the promoter of CNTF, and the recruitment of CREB and CREB-binding protein to the CNTF promoter by aspirin suggest that aspirin increases the expression of the Cntf gene via the activation of CREB. Furthermore, we demonstrate that aspirin-induced astroglial CNTF was also functionally active and that supernatants of aspirin-treated astrocytes of wild type, but not Cntf null, mice increased myelin-associated proteins in oligodendrocytes and protected oligodendrocytes from TNF-α insult. These results highlight a new and novel myelinogenic property of aspirin, which may be of benefit for multiple sclerosis and other demyelinating disorders. PMID:23653362
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Talkhabi, Mahmood; Razavi, Seyed Morteza; Salari, Ali
2017-06-01
Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and quantitatively efficient direct cardiac reprogramming. Taken together, this study provides new insights into the complexity of cell fate conversion and better understanding of the roles of transcriptional activators, signaling pathways and protein kinases in increasing the efficiency of direct cardiac reprogramming and maturity of iCMs.
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Invited review: Regulation of skeletal muscle GLUT-4 expression by exercise.
Dohm, G Lynis
2002-08-01
The amount of GLUT-4 protein is a primary factor in determining the maximal rate of glucose transport into skeletal muscle. Therefore, it is important that we understand how exercise regulates GLUT-4 expression so that therapeutic strategies can be designed to increase muscle glucose disposal as a treatment for diabetes. Muscle contraction increases the rates of GLUT-4 transcription and translation. Transcriptional control likely requires at least two DNA binding proteins, myocyte enhancer factor-2 and GLUT-4 enhancer factor, which bind to the promoter. Increased GLUT-4 expression may be mediated by the enzyme AMP-activated kinase, which is activated during exercise and has been demonstrated to increase GLUT-4 transcription. Further research needs to be done to investigate whether AMP-activated kinase activates myocyte enhancer factor-2 and GLUT-4 enhancer factor to increase transcription of the GLUT-4 gene.
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Contact Activation of Blood Plasma and Factor XII by Ion-exchange Resins
Yeh, Chyi-Huey Josh; Dimachkie, Ziad O.; Golas, Avantika; Cheng, Alice; Parhi, Purnendu; Vogler, Erwin A.
2011-01-01
Sepharose ion-exchange particles bearing strong Lewis acid/base functional groups (sulfopropyl, carboxymethyl, quarternary ammonium, dimethyl aminoethyl, and iminodiacetic acid) exhibiting high plasma protein adsorbent capacities are shown to be more efficient activators of blood factor XII in neat-buffer solution than either hydrophilic clean-glass particles or hydrophobic octyl sepharose particles ( FXII→surfaceactivatorFXIIa; a.k.a autoactivation, where FXII is the zymogen and FXIIa is a procoagulant protease). In sharp contrast to the clean-glass standard of comparison, ion-exchange activators are shown to be inefficient activators of blood plasma coagulation. These contrasting activation properties are proposed to be due to the moderating effect of plasma-protein adsorption on plasma coagulation. Efficient adsorption of blood plasma proteins unrelated to the coagulation cascade impedes FXII contacts with ion-exchange particles immersed in plasma, reducing autoactivation, and causing sluggish plasma coagulation. By contrast, plasma proteins do not adsorb to hydrophilic clean glass and efficient autoactivation leads directly to efficient activation of plasma coagulation. It is also shown that competitive-protein adsorption can displace FXIIa adsorbed to the surface of ion-exchange resins. As a consequence of highly-efficient autoactivation and FXIIa displacement by plasma proteins, ion-exchange particles are slightly more efficient activators of plasma coagulation than hydrophobic octyl sepharose particles that do not bear strong Lewis acid/base surface functionalities but to which plasma proteins adsorb efficiently. Plasma proteins thus play a dual role in moderating contact activation of the plasma coagulation cascade. The principal role is impeding FXII contact with activating surfaces but this same effect can displace FXIIa from an activating surface into solution where the protease can potentiate subsequent steps of the plasma coagulation cascade. PMID:21982294
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Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian
2000-01-01
Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019
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Poppers, J; Mulvey, M; Khoo, D; Mohr, I
2000-12-01
Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.
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Stable factor; Proconvertin; Autoprothrombin I ... be caused by an abnormally low level of factor VII. ... Decreased factor VII activity may be related to: Deficiency of factor VII Disorder in which the proteins that control ...
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Labile factor; Proaccelerin; Ac-globulin ... be caused by an abnormally low level of factor V. ... Decreased factor V activity may be related to: Deficiency of factor V Disorder in which the proteins that control ...
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Kanda, Shigeru; Mochizuki, Yasushi; Miyata, Yasuyoshi; Kanetake, Hiroshi; Yamamoto, Nobuto
2002-09-04
The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.
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A Plasma Protein Indistinguishable from Ribosomal Protein S19
Semba, Umeko; Chen, Jun; Ota, Yoshihiko; Jia, Nan; Arima, Hidetoshi; Nishiura, Hiroshi; Yamamoto, Tetsuro
2010-01-01
A monocyte-chemoattracting factor is generated during blood coagulation and during clotting of platelet-rich plasma. This chemotactic factor attracts monocytes as a ligand of the C5a receptor; however, it inhibits C5a-induced neutrophil chemotaxis as an apparent receptor antagonist. The curious dual function of the serum monocyte chemotactic factor resembles that of the cross-linked homodimer of ribosomal protein S19 (RP S19). Indeed, the inactive precursor of the monocyte chemotactic factor was present in plasma, and the precursor molecule and RP S19, as well as the active form and the RP S19 dimer, were indistinguishable in terms of immunological reactivity and molecular size. Coagulation factor XIIIa, plasma transglutaminase, and membrane phosphatidylserine on the activated platelets were required for conversion of the precursor to the active form. In addition, the precursor molecule in plasma could be replaced by wild-type recombinant RP S19 but not by mutant forms of it. These results indicate that a molecule indistinguishable from RP S19 was present in plasma, and that the RP S19-like molecule was converted to the active form by a transglutaminase-catalyzed reaction on a scaffold that included the phosphatidylserine-exposed platelet membrane. PMID:20093496
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Kobayashi, Yuhko; Murata, Michiharu; Minami, Hideyuki; Yamamoto, Shuhei; Kagaya, Yasuaki; Hobo, Tokunori; Yamamoto, Akiko; Hattori, Tsukaho
2005-12-01
The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.
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Role of carbohydrate in multimeric structure of factor VIII/von Willebrand factor protein.
Gralnick, H R; Williams, S B; Rick, M E
1983-01-01
The carbohydrate moiety of the factor VIII/von Willebrand (vW) factor protein is important in the expression of vW factor activity and the intravascular survival of the protein. Studies of normal human factor VIII/vW factor protein indicate that there is a requirement of a full complement of penultimate galactose for the maintenance of a normal multimeric structure. Release of penultimate galactose by beta-galactosidase or modification by galactose oxidase results in loss of the largest molecular weight multimers and increased numbers of intermediate and smaller multimers. In contrast, terminal galactose on the factor VIII/vW factor protein does not appear to play a significant role in the maintenance of the multimer organization. The abnormalities in multimeric structure and molecular size were demonstrated by NaDodSO4/polyacrylamide/agarose gel electrophoresis, NaDodSO4/glyoxyl-agarose electrophoresis, and sucrose density ultracentrifugation. These studies indicate that the penultimate galactose plays a role in the maintenance of the largest multimers of the factor VIII/vW factor protein. This may explain why, in some patients with variant forms of vW disease, a carbohydrate abnormality also may affect the multimeric structure of the plasma factor VIII/vW factor protein. Images PMID:6601805
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Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R; Hellmann, Hanjo
2013-06-01
Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that cullin3-based E3 ligases have the potential to interact with a broad range of ethylene response factor (ERF)/APETALA2 (AP2) transcription factors, mediated by Math-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the wrinkled1 ERF/AP2 protein. Furthermore, loss of Math-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.
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Soond, Surinder M.; Terry, Jennifer L.; Colbert, Jeff D.; Riches, David W. H.
2003-01-01
We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Coimmunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-α. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-κB and increased NF-κB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS1-723) was found to inhibit NF-κB activation by TNF-α. Coprecipitation and coimmunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK. PMID:14585990
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Price, B D; Morris, J D; Hall, A
1989-01-01
The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase. PMID:2690829
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Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription.
Russo, M W; Sevetson, B R; Milbrandt, J
1995-01-01
NGFI-A (also called Egr1, Zif268, or Krox24) and the closely related proteins Krox20, NGFI-C, and Egr3 are zinc-finger transcription factors encoded by immediate-early genes which are induced by a wide variety of extracellular stimuli. NGFI-A has been implicated in cell proliferation, macrophage differentiation, synaptic activation, and long-term potentiation, whereas Krox20 is critical for proper hindbrain segmentation and peripheral nerve myelination. In previous work, a structure/function analysis of NGFI-A revealed a 34-aa inhibitory domain that was hypothesized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid system, we have isolated a cDNA clone which encodes a protein that interacts with this inhibitory domain and inhibits the ability of NGFI-A to activate transcription. This NGFI-A-binding protein, NAB1, is a 570-aa nuclear protein that bears no obvious sequence homology to known proteins. NAB1 also represses Krox20 activity, but it does not influence Egr3 or NGFI-G, thus providing a mechanism for the differential regulation of this family of immediate-early transcription factors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7624335
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A Systems Biology Approach to Link Nuclear Factor Kappa B Activation with Lethal Prostate Cancer
2014-05-01
developed as a routine clinical assay. 12 Task 1B: Perform protein profiling of circulating blood proteins and determine whether a protein...or set of proteins indicative of NFκB activation are associated with lethal prostate cancer. Circulating proteins will be assessed in two cohorts of...throughput functional genomic data. Nucleic acids research 2009;37:D885-90. 3. Parkinson H, Kapushesky M, Kolesnikov N, et al. ArrayExpress update--from
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Acting on Actin: Rac and Rho Played by Yersinia.
Aepfelbacher, Martin; Wolters, Manuel
2017-01-01
Pathogenic bacteria of the genus Yersinia include Y. pestis-the agent of plaque-and two enteropathogens, Y. enterocolitica, and Y. pseudotuberculosis. These pathogens have developed an array of virulence factors aimed at manipulating Rho GTP-binding proteins and the actin cytoskeleton in host cells to cross the intestinal barrier and suppress the immune system. Yersinia virulence factors include outer membrane proteins triggering cell invasion by binding to integrins, effector proteins injected into host cells to manipulate Rho protein functions and a Rho protein-activating exotoxin. Here, we present an overview of how Yersinia and host factors are integrated in a regulatory network that orchestrates the subversion of host defense.
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Omar, M N; Shouk, T A; Khaleq, M A
1999-06-01
To examine the effect of medium molecular weight hydroxyethyl starch on protein C levels and the changes in the activation state of blood platelets, coagulation and fibrinolyis during and after 5 day of its infusion. Fifty male patients (mean age: 47 years, range 45-50 years) who required prostatectomy for benign prostatic hyperplasia were divided into two equal groups. One group was given 15 mL/kg body weight (mean volume 1000 mL +/- 100 mL) of 6% hydroxyethyl starch (HES) 200/0.5, the other received an equal volume of 5% human albumin during the operation. Blood samples were collected immediately before infusion (baseline values) and at 20, 40, 60, 90, 240, and 480 min after the infusion started then daily for the next 5 days postoperatively. Hematocrit, factor VIII:C, thrombin-antithrombin III complex; the anticoagulant protein C levels; the fibrinolytic parameters tissue type plasminogen activator (t-PA), and the fibrinolytic product D-Dimer and the platelet aggregation activity were measured. The data obtained did not detect any significant differences between HES and human albumin in the plasma levels of thrombin-antithrombin III complex, protein C, tissue-type plasminogen activator and the fibrin split products D-Dimer. Factor VIII:C and platelet aggregation were significantly lower in the hydroxyethyl starch group in comparison with albumin. Baseline values were attained postoperatively for factor VIII:C and platelet aggregation by the first and fifth days, respectively. The lowering effect of medium molecular weight hydroxyethyl starch on factor VIII:C would not be attributed to increased proteolytic activity of protein C on this coagulation cofactor because there is a nonsignificant change in protein C levels.
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NASA Technical Reports Server (NTRS)
Biermann, B. J.; Pao, L. I.; Feldman, L. J.
1994-01-01
Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.
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Donovan, Marissa R; Marr, Michael T
2016-09-02
Maintaining protein homeostasis is critical for survival at the cellular and organismal level (Morimoto, R. I. (2011) Cold Spring Harb. Symp. Quant. Biol. 76, 91-99). Cells express a family of molecular chaperones, the heat shock proteins, during times of oxidative stress to protect against proteotoxicity. We have identified a second stress responsive transcription factor, dFOXO, that works alongside the heat shock transcription factor to activate transcription of both the small heat shock protein and the large heat shock protein genes. This expression likely protects cells from protein misfolding associated with oxidative stress. Here we identify the regions of the Hsp70 promoter essential for FOXO-dependent transcription using in vitro methods and find a physiological role for FOXO-dependent expression of heat shock proteins in vivo. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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NASA Astrophysics Data System (ADS)
Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.
2000-05-01
RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.
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Jaiswal, Mamta; Dvorsky, Radovan; Ahmadian, Mohammad Reza
2013-02-08
The diffuse B-cell lymphoma (Dbl) family of the guanine nucleotide exchange factors is a direct activator of the Rho family proteins. The Rho family proteins are involved in almost every cellular process that ranges from fundamental (e.g. the establishment of cell polarity) to highly specialized processes (e.g. the contraction of vascular smooth muscle cells). Abnormal activation of the Rho proteins is known to play a crucial role in cancer, infectious and cognitive disorders, and cardiovascular diseases. However, the existence of 74 Dbl proteins and 25 Rho-related proteins in humans, which are largely uncharacterized, has led to increasing complexity in identifying specific upstream pathways. Thus, we comprehensively investigated sequence-structure-function-property relationships of 21 representatives of the Dbl protein family regarding their specificities and activities toward 12 Rho family proteins. The meta-analysis approach provides an unprecedented opportunity to broadly profile functional properties of Dbl family proteins, including catalytic efficiency, substrate selectivity, and signaling specificity. Our analysis has provided novel insights into the following: (i) understanding of the relative differences of various Rho protein members in nucleotide exchange; (ii) comparing and defining individual and overall guanine nucleotide exchange factor activities of a large representative set of the Dbl proteins toward 12 Rho proteins; (iii) grouping the Dbl family into functionally distinct categories based on both their catalytic efficiencies and their sequence-structural relationships; (iv) identifying conserved amino acids as fingerprints of the Dbl and Rho protein interaction; and (v) defining amino acid sequences conserved within, but not between, Dbl subfamilies. Therefore, the characteristics of such specificity-determining residues identified the regions or clusters conserved within the Dbl subfamilies.
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Single-Molecule Studies of Actin Assembly and Disassembly Factors
Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.
2014-01-01
The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103
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Lai, Char-Chang; Edwards, Anne P B; DiMaio, Daniel
2005-02-01
The bovine papillomavirus E5 protein is a 44-amino-acid transmembrane protein that transforms cells by binding to the transmembrane region of the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in sustained receptor signaling. However, there are published reports that certain mutants with amino acid substitutions in the membrane-spanning segment of the E5 protein transform cells without activating the PDGF beta receptor. We re-examined several of these transmembrane mutants, and here we present five lines of evidence that these mutants do in fact activate the PDGF beta receptor, resulting in cellular signaling and transformation.
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Endogenous versus Exogenous Growth Factor Regulation of Articular Chondrocytes
Shi, Shuiliang; Chan, Albert G.; Mercer, Scott; Eckert, George J.; Trippel, Stephen B.
2014-01-01
Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF-I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF-ß1 stimulated these reparative functions, while endogenous TGF-ß1 had little effect. Endogenous TGF-ß1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF-I, gene therapy or protein therapy appear to be viable options. In contrast, TGF-ß1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form. PMID:24105960
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Bae, Jong-Sup; Yang, Likui; Rezaie, Alireza R
2010-11-05
We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.
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Shyu, Kou-Gi; Cheng, Wen-Pin; Wang, Bao-Wei; Chang, Hang
2014-03-01
The expression of MURC (muscle-restricted coiled-coil protein), a hypertrophy-regulated gene, increases during pressure overload. Hypoxia can cause myocardial hypertrophy; however, how hypoxia affects the regulation of MURC in cardiomyocytes undergoing hypertrophy is still unknown. The aim of the present study was to test the hypothesis that hypoxia induces MURC expression in cardiomyocytes during hypertrophy. The expression of MURC was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia and in an in vivo model of AMI (acute myocardial infarction) to induce myocardial hypoxia in adult rats. MURC protein and mRNA expression were significantly enhanced by hypoxia. MURC proteins induced by hypoxia were significantly blocked after the addition of PD98059 or ERK (extracellular-signal-regulated kinase) siRNA 30 min before hypoxia. Gel-shift assay showed increased DNA-binding activity of SRF (serum response factor) after hypoxia. PD98059, ERK siRNA and an anti-TGF-β (transforming growth factor-β) antibody abolished the SRF-binding activity enhanced by hypoxia or exogenous administration of TGF-β. A luciferase promoter assay demonstrated increased transcriptional activity of SRF in cardiomyocytes by hypoxia. Increased βMHC (β-myosin heavy chain) and BNP (B-type natriuretic peptide) protein expression and increased protein synthesis was identified after hypoxia with the presence of MURC in hypertrophic cardiomyocytes. MURC siRNA inhibited the hypertrophic marker protein expression and protein synthesis induced by hypoxia. AMI in adult rats also demonstrated increased MURC protein expression in the left ventricular myocardium. In conclusion, hypoxia in cultured rat neonatal cardiomyocytes increased MURC expression via the induction of TGF-β, SRF and the ERK pathway. These findings suggest that MURC plays a role in hypoxia-induced hypertrophy in cardiomyocytes.
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WFIKKN1 and WFIKKN2: "Companion" proteins regulating TGFB activity.
Monestier, Olivier; Blanquet, Véronique
2016-12-01
The WFIKKN (WAP, Follistatin/kazal, Immunoglobulin, Kunitz and Netrin domain-containing) protein family is composed of two multidomain proteins: WFIKKN1 and WFIKKN2. They were formed by domain shuffling and are likely present in deuterostoms. The WFIKKN (also called GASP) proteins are well known for their function in muscle and skeletal tissues, namely, inhibition of certain members of the transforming growth factor beta (TGFB) superfamily such as myostatin (MSTN) and growth and differentiation factor 11 (GDF11). However, the role of the WFIKKN proteins in other tissues is still poorly understood in spite of evidence suggesting possible action in the inner ear, brain and reproduction. Further, several recent studies based on next generation technologies revealed differential expression of WFIKKN1 and WFIKKN2 in various tissues suggesting that their function is not limited to MSTN and GDF11 inhibition in musculoskeletal tissue. In this review, we summarize current knowledge about the WFIKKN proteins and propose that they are "companion" proteins for various growth factors by providing localized and sustained presentation of TGFB proteins to their respective receptors, thus regulating the balance between the activation of Smad and non-Smad pathways by TGFB. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Amiral, Jean; Vissac, Anne Marie; Seghatchian, Jerard
2017-12-01
Activated Protein C Resistance is mainly associated to a factor V mutation (RQ506), which induces a deficient inactivation of activated factor V by activated protein C, and is associated to an increased risk of venous and arterial thrombosis in affected individuals, caused by the prolonged activated factor V survival. Its prevalence is mainly in Caucasians (about 5%), and this mutation is absent in Africans and Asians. Presence of Factor V-Leiden is usually evidenced with clotting methods, using a two-step APTT assay performed without or with APC: prolongation of blood coagulation time is decreased if this factor is present. The R506Q Factor V-Leiden mutation is now usually characterized using molecular biology, and this technique tends to become the first intention assay for characterization of patients. Both techniques are qualitative, and allow classifying tested individuals as heterozygotes or homozygotes for the mutation, when present. A new quantitative assay for Factor V-Leiden, using a one-step clotting method, has been developed, and designed with highly purified human coagulation proteins. Clotting is triggered with human Factor Xa, in presence of calcium and phospholipids (mixture which favours APC action over clotting process). Diluted tested plasma, is supplemented with a clotting mixture containing human fibrinogen, prothrombin, and protein S at a constant concentration. APC is added, and clotting is initiated with calcium. Calibration is performed with a pool of plasmas from patients carrying the R506Q Factor V mutation, and its mixtures with normal plasma. Homozygous patients have clotting times of about <40sec; heterozygous patients have clotting times of about 40-60sec and normal individuals yield clotting times >70sec. Factor V-Leiden concentration is usually >75% in homozygous patients, 30-60% in heterozygous patients and below 5% in normal. The assay is insensitive to clotting factor deficiencies (II, VII, VIII: C, IX, X), dicoumarol or heparin therapies, and has no interference with lupus anticoagulant (LA). This new assay for Factor V-Leiden can be easily used in any coagulation laboratory, is performed as a single test, and is quantitative. This assay has a high robustness, is accurate and presents a good intra- (<3%) and inter-assay (<5%) variability. It contributes solving most of the laboratory issues faced when testing factor V-Leiden. Quantitation of Factor V-L could contribute to a better assessment of thrombotic risk in affected patients, as this complication is first associated to and caused by the presence of a defined amount of FVa. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Welle, Stephen; Burgess, Kerri; Mehta, Sangeeta
2009-03-01
Knocking out myostatin activity during development increases the rate of muscle protein synthesis. The present study was done to determine whether postdevelopmental loss of myostatin activity stimulates myofibrillar protein synthesis and the phosphorylation of some of the proteins involved in regulation of protein synthesis rate. Myostatin activity was inhibited for 4 days, in 4- to 5-mo-old male mice, with injections of an anti-myostatin antibody (JA16). The mean myofibrillar synthesis rate increased 19% (P < 0.01) relative to the mean rate in saline-treated mice, as determined by incorporation of deuterium-labeled phenylalanine. JA16 increased phosphorylation of p70 S6 kinase (S6K) and ribosomal protein S6 (rpS6) 1.9-fold (P < 0.05). It did not affect phosphorylation of eukaryotic initiation factor 4E-binding protein-1 or Akt. Microarrays and real-time PCR analyses indicated that JA16 administration did not selectively enrich levels of mRNAs encoding myofibrillar proteins, ribosomal proteins, or translation initiation and elongation factors. Rapamycin treatment did not affect the rate of myofibrillar protein synthesis whether or not the mice received JA16 injections, although it eliminated the phosphorylation of S6K and rpS6. We conclude that the normal level of myostatin activity in mature muscle is sufficient to inhibit myofibrillar synthesis rate and phosphorylation of S6K and rpS6. Reversal of the inhibition of myofibrillar synthesis with an anti-myostatin antibody is not dependent on mTOR activation.
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Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon
2012-10-26
The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.
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Choli-Papadopoulou, Theodora; Kottakis, Filippos; Papadopoulos, Georgios; Pendas, Stefanos
2011-06-07
Helicobacter pylori (H. pylori) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer. Within this work we present the implication of C-terminal region of H. pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evidence that the C-terminal region of H. pylori activating protein is indispensable for neutrophil adhesion to endothelial cells, a step necessary to H. pylori inflammation. In addition we show that arabino galactan proteins derived from chios mastic gum, the natural resin of the plant Pistacia lentiscus var. Chia inhibit neutrophil activation in vitro.
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Drosophila Spire is an actin nucleation factor.
Quinlan, Margot E; Heuser, John E; Kerkhoff, Eugen; Mullins, R Dyche
2005-01-27
The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.
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Growth factors and hormones pro-peptides: the unexpected adventures of the BDNF prodomain.
Zanin, Juan Pablo; Unsain, Nicolás; Anastasia, Agustin
2017-05-01
Most growth factors and hormones are synthesized as pre-pro-proteins which are processed to the biologically active mature protein. The pre- and prodomains are cleaved from the precursor protein in the secretory pathway or, in some cases, extracellularly. The canonical functions of these prodomains are to assist in folding and stabilization of the mature domain, to direct intra and extracellular localization, to facilitate storage, and to regulate bioavailability of their mature counterpart. Recently, exciting evidence has revealed that prodomains of certain growth factors, after cleaved from the precursor pro-protein, can act as independent active signaling molecules. In this review, we discuss the various classical functions of prodomains, and the biological consequences of these pro-peptides acting as ligands. We will focus our attention on the brain-derived neurotrophic factor prodomain (pBDNF), which has been recently described as a novel secreted ligand influencing neuronal morphology and physiology. © 2017 International Society for Neurochemistry.
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Bioglass Activated Skin Tissue Engineering Constructs for Wound Healing.
Yu, Hongfei; Peng, Jinliang; Xu, Yuhong; Chang, Jiang; Li, Haiyan
2016-01-13
Wound healing is a complicated process, and fibroblast is a major cell type that participates in the process. Recent studies have shown that bioglass (BG) can stimulate fibroblasts to secrete a multitude of growth factors that are critical for wound healing. Therefore, we hypothesize that BG can stimulate fibroblasts to have a higher bioactivity by secreting more bioactive growth factors and proteins as compared to untreated fibroblasts, and we aim to construct a bioactive skin tissue engineering graft for wound healing by using BG activated fibroblast sheet. Thus, the effects of BG on fibroblast behaviors were studied, and the bioactive skin tissue engineering grafts containing BG activated fibroblasts were applied to repair the full skin lesions on nude mouse. Results showed that BG stimulated fibroblasts to express some critical growth factors and important proteins including vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, collagen I, and fibronectin. In vivo results revealed that fibroblasts in the bioactive skin tissue engineering grafts migrated into wound bed, and the migration ability of fibroblasts was stimulated by BG. In addition, the bioactive BG activated fibroblast skin tissue engineering grafts could largely increase the blood vessel formation, enhance the production of collagen I, and stimulate the differentiation of fibroblasts into myofibroblasts in the wound site, which would finally accelerate wound healing. This study demonstrates that the BG activated skin tissue engineering grafts contain more critical growth factors and extracellular matrix proteins that are beneficial for wound healing as compared to untreated fibroblast cell sheets.
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Endurance Factors Improve Hippocampal Neurogenesis and Spatial Memory in Mice
ERIC Educational Resources Information Center
Kobilo, Tali; Yuan, Chunyan; van Praag, Henriette
2011-01-01
Physical activity improves learning and hippocampal neurogenesis. It is unknown whether compounds that increase endurance in muscle also enhance cognition. We investigated the effects of endurance factors, peroxisome proliferator-activated receptor [delta] agonist GW501516 and AICAR, activator of AMP-activated protein kinase on memory and…
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Zhang, Wei; Qiao, Haishi; Lv, Yuanzi; Wang, Jingjing; Chen, Xiaoqing; Hou, Yayi; Tan, Renxiang; Li, Erguang
2014-01-01
Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC50 value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC50 was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5′-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication. PMID:25330384
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The MAP kinase pathway is involved in odontoblast stimulation via p38 phosphorylation.
Simon, Stephane; Smith, Anthony J; Berdal, Ariane; Lumley, Philip J; Cooper, Paul R
2010-02-01
We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when odontoblasts are stimulated such as during tertiary reactionary dentinogenesis. We stimulated immortalized, odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-beta1, tumor necrosis factor-alpha (TNF-alpha), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation. Our results suggest that the p38-MAPKinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation. Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis. Copyright 2010 American Association of Endodontists. All rights reserved.
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Khan, Shamila; Cole, Nerida; Hume, Emma B H; Garthwaite, Linda L; Nguyen-Khuong, Terry; Walsh, Bradley J; Willcox, Mark D P
2016-10-01
Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Fang, Ying; Fang, Liurong; Wang, Yang; Lei, Yingying; Luo, Rui; Wang, Dang; Chen, Huanchun; Xiao, Shaobo
2012-04-30
Nuclear factor-kappaB (NF-κB) is an inducible transcription factor that plays a key role in inflammation and immune responses, as well as in the regulation of cell proliferation and survival. Previous studies by our group and others have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection could activate NF-κB in MARC-145 cells and alveolar macrophages. The nucleocapsid (N) protein was identified as an NF-κB activator among the structural proteins encoded by PRRSV; however, it remains unclear whether the nonstructural proteins (Nsps) contribute to NF-κB activation. In this study, we identified which Nsps can activate NF-κB and investigated the potential mechanism(s) by which they act. By screening the individual Nsps of PRRSV strain WUH3, Nsp2 exhibited great potential to activate NF-κB in MARC-145 and HeLa cells. Overexpression of Nsp2 induced IκBα degradation and nuclear translocation of NF-κB. Furthermore, Nsp2 also induced NF-κB-dependent inflammatory factors, including interleukin (IL)-6, IL-8, COX-2, and RANTES. Compared with the Nsp2 of the classical PRRSV strain, the Nsp2 of highly pathogenic PRRSV (HP-PRRSV) strains that possess a 30 amino acid (aa) deletion in Nsp2 displayed greater NF-κB activation. However, the 30-aa deletion was demonstrated to not be associated with NF-κB activation. Further functional domain analyses revealed that the hypervariable region (HV) of Nsp2 was essential for NF-κB activation. Taken together, these data indicate that PRRSV Nsp2 is a multifunctional protein participating in the modulation of host inflammatory response, which suggests an important role of Nsp2 in pathogenesis and disease outcomes.
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Che, Karlhans Fru; Shankar, Esaki Muthu; Muthu, Sundaram; Zandi, Sasan; Sigvardsson, Mikael; Hinkula, Jorma; Messmer, Davorka; Larsson, Marie
2012-01-01
Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1–primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules. PMID:22777388
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Chen, Angela; Huang, Yan Chang; Wang, Pin Yao; Kemp, Sadie E.
2012-01-01
During development, proneural transcription factors of the basic helix-loop-helix (bHLH) family are required to commit cells to a neural fate. In Drosophila neurogenesis, a key mechanism promoting sense organ precursor (SOP) fate is the synergy between proneural factors and their coactivator Senseless in transcriptional activation of target genes. Here we present evidence that posttranslational modification by SUMO enhances this synergy via an effect on Senseless protein. We show that Senseless is a direct target for SUMO modification and that mutagenesis of a predicted SUMOylation motif in Senseless reduces Senseless/proneural synergy both in vivo and in cell culture. We propose that SUMOylation of Senseless via lysine 509 promotes its synergy with proneural proteins during transcriptional activation and hence regulates an important step in neurogenesis leading to the formation and maturation of the SOPs. PMID:22586269
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Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains*
Kienast, Yvonne; Jucknischke, Ute; Scheiblich, Stefan; Thier, Martina; de Wouters, Mariana; Haas, Alexander; Lehmann, Christian; Brand, Verena; Bernicke, Dirk; Honold, Konrad; Lorenz, Stefan
2016-01-01
By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants. PMID:26677222
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Damania, Blossom; Mital, Renu; Alwine, James C.
1998-01-01
The TATA-binding protein (TBP) is common to the basal transcription factors of all three RNA polymerases, being associated with polymerase-specific TBP-associated factors (TAFs). Simian virus 40 large T antigen has previously been shown to interact with the TBP-TAFII complexes, TFIID (B. Damania and J. C. Alwine, Genes Dev. 10:1369–1381, 1996), and the TBP-TAFI complex, SL1 (W. Zhai, J. Tuan, and L. Comai, Genes Dev. 11:1605–1617, 1997), and in both cases these interactions are critical for transcriptional activation. We show a similar mechanism for activation of the class 3 polymerase III (pol III) promoter for the U6 RNA gene. Large T antigen can activate this promoter, which contains a TATA box and an upstream proximal sequence element but cannot activate the TATA-less, intragenic VAI promoter (a class 2, pol III promoter). Mutants of large T antigen that cannot activate pol II promoters also fail to activate the U6 promoter. We provide evidence that large T antigen can interact with the TBP-containing pol III transcription factor human TFIIB-related factor (hBRF), as well as with at least two of the three TAFs in the pol III-specific small nuclear RNA-activating protein complex (SNAPc). In addition, we demonstrate that large T antigen can cofractionate and coimmunoprecipitate with the hBRF-containing complex TFIIIB derived from HeLa cells infected with a recombinant adenovirus which expresses large T antigen. Hence, similar to its function with pol I and pol II promoters, large T antigen interacts with TBP-containing, basal pol III transcription factors and appears to perform a TAF-like function. PMID:9488448
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Li, Zhiqiang; Shu, Qingming; Li, Lingzhi; Ge, Maolin; Zhang, Yongliang
2014-01-01
Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott's method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury. PMID:25206921
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Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A
2003-01-01
Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173
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Regulatory role of the 90-kDa-heat-shock protein (Hsp90) and associated factors on gene expression.
Erlejman, Alejandra G; Lagadari, Mariana; Toneatto, Judith; Piwien-Pilipuk, Graciela; Galigniana, Mario D
2014-02-01
The term molecular chaperone was first used to describe the ability of nucleoplasmin to prevent the aggregation of histones with DNA during the assembly of nucleosomes. Subsequently, the name was extended to proteins that mediate the post-translational assembly of oligomeric complexes protecting them from denaturation and/or aggregation. Hsp90 is a 90-kDa molecular chaperone that represents the major soluble protein of the cell. In contrast to most conventional chaperones, Hsp90 functions as a refined sensor of protein function and its principal role in the cell is to facilitate biological activity to properly folded client proteins that already have a preserved tertiary structure. Consequently, Hsp90 is related to basic cell functions such as cytoplasmic transport of soluble proteins, translocation of client proteins to organelles, and regulation of the biological activity of key signaling factors such as protein kinases, ubiquitin ligases, steroid receptors, cell cycle regulators, and transcription factors. A growing amount of evidence links the protective action of this molecular chaperone to mechanisms related to posttranslational modifications of soluble nuclear factors as well as histones. In this article, we discuss some aspects of the regulatory action of Hsp90 on transcriptional regulation and how this effect could have impacted genetic assimilation mechanism in some organisms. Copyright © 2013 Elsevier B.V. All rights reserved.
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Snow, Wanda M.; Pahlavan, Payam S.; Djordjevic, Jelena; McAllister, Danielle; Platt, Eric E.; Alashmali, Shoug; Bernstein, Michael J.; Suh, Miyoung; Albensi, Benedict C.
2015-01-01
Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory. PMID:26635523
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GIV/Girdin transmits signals from multiple receptors by triggering trimeric G protein activation.
Garcia-Marcos, Mikel; Ghosh, Pradipta; Farquhar, Marilyn G
2015-03-13
Activation of trimeric G proteins has been traditionally viewed as the exclusive job of G protein-coupled receptors (GPCRs). This view has been challenged by the discovery of non-receptor activators of trimeric G proteins. Among them, GIV (a.k.a. Girdin) is the first for which a guanine nucleotide exchange factor (GEF) activity has been unequivocally associated with a well defined motif. Here we discuss how GIV assembles alternative signaling pathways by sensing cues from various classes of surface receptors and relaying them via G protein activation. We also describe the dysregulation of this mechanism in disease and how its targeting holds promise for novel therapeutics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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A Systems Biology Approach to Link Nuclear Factor Kappa B Activation with Lethal Prostate Cancer
2013-05-01
developed as a routine clinical assay. Task 1B: Perform protein profiling of circulating blood proteins and determine whether a protein or set of...proteins indicative of NFκB activation are associated with lethal prostate cancer. Circulating proteins will be assessed in two cohorts of 312...functional genomic data. Nucleic Acids Res 2009;37:D885-90. 3. Parkinson H, Kapushesky M, Kolesnikov N, et al. ArrayExpress update--from an archive of
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Bognar, Eszter; Sarszegi, Zsolt; Szabo, Aliz; Debreceni, Balazs; Kalman, Nikoletta; Tucsek, Zsuzsanna; Sumegi, Balazs; Gallyas, Ferenc
2013-01-01
Background Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways. Methods & Findings The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation. Conclusions These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease. PMID:23755222
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N-(4-methoxyphenyl) caffeamide-induced melanogenesis inhibition mechanisms.
Kuo, Yueh-Hsiung; Chen, Chien-Chia; Wu, Po-Yuan; Wu, Chin-Sheng; Sung, Ping-Jyun; Lin, Chien-Yih; Chiang, Hsiu-Mei
2017-01-23
The derivative of caffeamide exhibits antioxidant and antityrosinase activity. The activity and mechanism of N-(4-methoxyphenyl) caffeamide (K36E) on melanogenesis was investigated. B16F0 cells were treated with various concentrations of K36E; the melanin contents and related signal transduction were studied. Western blotting assay was applied to determine the protein expression, and spectrophotometry was performed to identify the tyrosinase activity and melanin content. Our results indicated that K36E reduced α-melanocyte-stimulating hormone (α-MSH)-induced melanin content and tyrosinase activity in B16F0 cells. In addition, K36E inhibited the expression of phospho-cyclic adenosine monophosphate (cAMP)-response element-binding protein, microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1). K36E activated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3 beta (GSK3β), leading to the inhibition of MITF transcription activity. K36E attenuated α-MSH induced cAMP pathways, contributing to hypopigmentation. K36E regulated melanin synthesis through reducing the expression of downstream proteins including p-CREB, p-AKT, p-GSK3β, tyrosinase, and TRP-1, and activated the transcription factor, MITF. K36E may have the potential to be developed as a skin whitening agent.
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Coronavirus virulence genes with main focus on SARS-CoV envelope gene.
DeDiego, Marta L; Nieto-Torres, Jose L; Jimenez-Guardeño, Jose M; Regla-Nava, Jose A; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Usera, Fernando; Enjuanes, Luis
2014-12-19
Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and -7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus β CoVs such as MERS-CoV), but not in others (genus β CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal models and in humans. The modification or deletion of different motifs within E protein, including the transmembrane domain that harbors an ion channel activity, small sequences within the middle region of the carboxy-terminus of E protein, and its most carboxy-terminal end, which contains a PDZ domain-binding motif (PBM), is sufficient to attenuate the virus. Interestingly, a comprehensive collection of SARS-CoVs in which these motifs have been modified elicited full and long-term protection even in old mice, making those deletion mutants promising vaccine candidates. These data indicate that despite its small size, E protein drastically influences the replication of CoVs and their pathogenicity. Although E protein is not essential for CoV genome replication or subgenomic mRNA synthesis, it affects virus morphogenesis, budding, assembly, intracellular trafficking, and virulence. In fact, E protein is responsible in a significant proportion of the inflammasome activation and the associated inflammation elicited by SARS-CoV in the lung parenchyma. This exacerbated inflammation causes edema accumulation leading to acute respiratory distress syndrome (ARDS) and, frequently, to the death of infected animal models or human patients. Copyright © 2014 Elsevier B.V. All rights reserved.
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CORONAVIRUS VIRULENCE GENES WITH MAIN FOCUS ON SARS-CoV ENVELOPE GENE
DeDiego, Marta L.; Nieto-Torres, Jose L.; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Usera, Fernando; Enjuanes, Luis
2014-01-01
Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and 7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus β CoVs such as MERS-CoV), but not in others (genus β CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal models and in humans. The modification or deletion of different motifs within E protein, including the transmembrane domain that harbors an ion channel activity, small sequences within the middle region of the carboxy-terminus of E protein, and its most carboxy-terminal end, which contains a PDZ domain-binding motif (PBM) is sufficient to attenuate the virus. Interestingly, a comprehensive collection of SARS-CoVs in which these motifs have been modified elicited full and long-term protection even in old mice, making those deletion mutants promising vaccine candidates. These data indicate that despite its small size, E protein drastically influences the replication of CoVs and their pathogenicity. Although E protein is not essential for CoV genome replication or subgenomic mRNA synthesis, it affects virus morphogenesis, budding, assembly, intracellular trafficking, and virulence. In fact, E protein is responsible in a significant proportion of the inflammasome activation and the associated inflammation elicited by SARS-CoV in the lung parenchyma. This exacerbated inflammation causes edema accumulation leading to acute respiratory distress syndrome (ARDS) and, frequently, to the death of infected animal models or human patients. PMID:25093995
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Regulation of cAMP on the first mitotic cell cycle of mouse embryos.
Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi
2008-03-01
Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.
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Wang, Xianmiao; Li, Ying; Mao, Aiping; Li, Chao; Li, Yongkui; Tien, Po
2010-09-01
Viral RNAs produced during viral infection are recognized by the cytoplasmic RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). A central adapter protein downstream of RIG-I and MDA5 is the mitochondrial membrane protein virus-induced signaling adaptor (VISA), which mediates the induction of type I interferons (IFNs) through the activation of transcription factors such as nuclear factor-kappaB (NF-kappaB) and IFN-regulatory factor-3 (IRF3). Here we found that hepatitis B virus (HBV)-encoded X protein (HBx) acts as an inhibitor of virus-triggered IRF3 activation and IFN-beta induction. Reporter and plaque assays indicate that HBx inhibits signaling by components upstream but not downstream of VISA. Immunoprecipitation experiments indicate that HBx interacts with VISA and disrupts the association of VISA with its upstream and downstream components. These findings suggest that HBx acts as a suppressor of virus-triggered induction of type I IFNs, which explains the observation that HBV causes transient and chronic infection in hepatocytes but fails to activate the pattern recognition receptor-mediated IFN induction pathways.
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Li, Jingxia; Zhang, Dongyun; Stoner, Gary D; Huang, Chuanshu
2008-04-01
The chemopreventive properties of edible berries have been demonstrated both in vitro and in vivo, however, the specific molecular mechanisms underlying their anti-cancer effects are largely unknown. Our previous studies have shown that a methanol extract fraction of freeze-dried black raspberries inhibits benzoapyrene (BaP)-induced transformation of Syrian hamster embryo cells. This fraction also blocks activation of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) induced by benzoapyrene diol-epoxide (BaPDE) in mouse epidermal JB6 Cl 41 cells. To determine if different berry types exhibit specific mechanisms for their anti-cancer effects, we compared the effects of extract fractions from both black raspberries and strawberries on BaPDE-induced activation of various signaling pathways in Cl 41 cells. Black raspberry fractions inhibited the activation of AP-1, NF-kappaB, and nuclear factor of activated T cells (NFAT) by BaPDE as well as their upstream PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. In contrast, strawberry fractions inhibited NFAT activation, but did not inhibit the activation of AP-1, NF-kappaB or the PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. Consistent with the effects on NFAT activation, tumor necrosis factor-alpha (TNF-alpha) induction by BaPDE was blocked by extract fractions of both black raspberries and strawberries, whereas vascular endothelial growth factor (VEGF) expression, which depends on AP-1 activation, was suppressed by black raspberry fractions but not strawberry fractions. These results suggest that black raspberry and strawberry components may target different signaling pathways in exerting their anti-carcinogenic effects. (c) 2007 Wiley-Liss, Inc.
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Li, Jingxia; Zhang, Dongyun; Stoner, Gary D.; Huang, Chuanshu
2013-01-01
The chemopreventive properties of edible berries have been demonstrated both in vitro and in vivo, however, the specific molecular mechanisms underlying their anti-cancer effects are largely unknown. Our previous studies have shown that a methanol extract fraction of freeze-dried black raspberries inhibits benzoapyrene (BaP)-induced transformation of Syrian hamster embryo cells. This fraction also blocks activation of activator protein-1 (AP-1) and nuclear factor κB (NF-κB) induced by benzoapyrene diol-epoxide (BaPDE) in mouse epidermal JB6 Cl 41 cells. To determine if different berry types exhibit specific mechanisms for their anti-cancer effects, we compared the effects of extract fractions from both black raspberries and strawberries on BaPDE-induced activation of various signaling pathways in Cl 41 cells. Black raspberry fractions inhibited the activation of AP-1, NF-κB, and nuclear factor of activated T cells (NFAT) by BaPDE as well as their upstream PI-3K/Akt-p70S6K and mitogen-activated protein kinase pathways. In contrast, strawberry fractions inhibited NFAT activation, but did not inhibit the activation of AP-1, NF-κB or the PI-3K/Akt-p70S6K and mitogen-activated protein kinase pathways. Consistent with the effects on NFAT activation, tumor necrosis factor-α (TNF-α) induction by BaPDE was blocked by extract fractions of both black raspberries and strawberries, whereas vascular endothelial growth factor (VEGF) expression, which depends on AP-1 activation, was suppressed by black raspberry fractions but not strawberry fractions. These results suggest that black raspberry and strawberry components may target different signaling pathways in exerting their anti-carcinogenic effects. PMID:18085529
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Adaptive and regulatory mechanisms in aged rats with postoperative cognitive dysfunction
Bi, Yanlin; Liu, Shuyun; Yu, Xinjuan; Wang, Mingshan; Wang, Yuelan
2014-01-01
Inflammation may play a role in postoperative cognitive dysfunction. 5′ Adenosine monophosphate-activated protein kinase, nuclear factor-kappa B, interleukin-1β, and tumor necrosis factor-α are involved in inflammation. Therefore, these inflammatory mediators may be involved in postoperative cognitive dysfunction. Western immunoblot analysis revealed 5′ adenosine monophosphate-activated protein kinase and nuclear factor-kappa B in the hippocampus of aged rats were increased 1–7 days after splenectomy. Moreover, interleukin-1β and tumor necrosis factor-α were upregulated and gradually decreased. Therefore, these inflammatory mediators may participate in the splenectomy model of postoperative cognitive dysfunction in aged rats. PMID:25206851
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A ribosome-dependent GTPase from yeast distinct from elongation factor 2.
Skogerson, L; Wakatama, E
1976-01-01
Three proteins required for poly(U)-directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2. PMID:174100
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Wecke, Tina; Halang, Petra; Staroń, Anna; Dufour, Yann S; Donohue, Timothy J; Mascher, Thorsten
2012-01-01
Bacteria need signal transducing systems to respond to environmental changes. Next to one- and two-component systems, alternative σ factors of the extra-cytoplasmic function (ECF) protein family represent the third fundamental mechanism of bacterial signal transduction. A comprehensive classification of these proteins identified more than 40 phylogenetically distinct groups, most of which are not experimentally investigated. Here, we present the characterization of such a group with unique features, termed ECF41. Among analyzed bacterial genomes, ECF41 σ factors are widely distributed with about 400 proteins from 10 different phyla. They lack obvious anti-σ factors that typically control activity of other ECF σ factors, but their structural genes are often predicted to be cotranscribed with carboxymuconolactone decarboxylases, oxidoreductases, or epimerases based on genomic context conservation. We demonstrate for Bacillus licheniformis and Rhodobacter sphaeroides that the corresponding genes are preceded by a highly conserved promoter motif and are the only detectable targets of ECF41-dependent gene regulation. In contrast to other ECF σ factors, proteins of group ECF41 contain a large C-terminal extension, which is crucial for σ factor activity. Our data demonstrate that ECF41 σ factors are regulated by a novel mechanism based on the presence of a fused regulatory domain. PMID:22950025
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Non-transcriptional interactions of Hox proteins: inventory, facts, and future directions.
Rezsohazy, René
2014-01-01
Hox proteins are conserved homeodomain transcription factors involved in the control of embryo patterning, organ development, and cell differentiation during animal development and adult life. Although recognizably active in gene regulation, accumulating reports support that Hox proteins are also active in controlling other molecular processes like mRNA translation, DNA repair, initiation of DNA replication, and possibly modulation of signal transduction. Here we review experimental evidence as well as databases entries indicative of non-transcriptional activities of Hox proteins. Copyright © 2013 Wiley Periodicals, Inc.
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Palmer, Steve; Groves, Nicola; Schindeler, Aaron; Yeoh, Thomas; Biben, Christine; Wang, Cheng-Chun; Sparrow, Duncan B.; Barnett, Louise; Jenkins, Nancy A.; Copeland, Neal G.; Koentgen, Frank; Mohun, Tim; Harvey, Richard P.
2001-01-01
We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic “myosacs.” The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer–binding factor (MEF)2, were also enhanced in an IGF-1 signaling–dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair. PMID:11381084
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Ostrovskaia, R U; Vakhitova, Iu V; Salimgareeva, M Kh; Iamidanov, R S; Sadovnikov, S V; Kapitsa, I G; Seredenin, S B
2010-12-01
The influence of noopept (N-phenylacetyl-L-prolylglycine ethyl ester, GVS-111)--a drug combining the nootrope and neuroprotector properties--on the activity of mitogen-activated protein kinases (MAPKs) and the level of NGF and BDNF gene and protein expression in the frontal cortex, hippocampus, and hypothalamus has been studied in rats. Under conditions of chronic administration (28 days, 0.5 mg/day, i.p.), noopept decreased the activity of stress-induced kinases (SAPK/JNK 46/54 and pERK1/2) in rat hippocampus and increases the level of mRNA of the BDNF gene in both hypothalamus and hippocampus. The content of BDNF protein in the hypothalamus was also somewhat increased. In the context of notions about the activation of stress-induced kinases, as an important factor of amyloidogenesis and tau-protein deposition in brain tissue, and the role of deficiency of the neurotrophic factors in the development of neurodegenerative processes, the observed decrease in the activity of stress-activated MAPKs and increased expression of BDNF as a result of noopept administration suggest thatthis drug hasaspecific activity withrespect to some pathogenetic mechanisms involved in the Alzheimer disease.
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Synergistic regulation of competence development in Bacillus subtilis by two Rap-Phr systems.
Bongiorni, Cristina; Ishikawa, Shu; Stephenson, Sophie; Ogasawara, Naotake; Perego, Marta
2005-07-01
The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Mol. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and RapF-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity.
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Gerber, AN; Wilson, CW; Li, Y-J; Chuang, P-T
2012-01-01
The mechanism by which activation of the Hedgehog (Hh) pathway modulates differentiation and promotes oncogenesis in specific tissues is poorly understood. We therefore, analysed rhabdomyosarcomas from mice that were haploinsufficient for the Hh-binding protein, Hip1, or for the Hh receptor, Patched 1 (Ptch1). Transfection of the Hh-regulated transcription factor Gli1, which is expressed in a subset of mouse and human rhabdomyosarcomas, suppressed differentiation of myogenic rhabdomyosarcoma lines generated from Hip1+/− and Ptch1+/− mice. The closely related factor, Gli2, had similar effects. Gli1 and Gli2 inhibited myogenesis by repressing the capacity of MyoD to activate transcription. Deletion analysis of Gli1 indicated that multiple domains of Gli1 are required for efficient inhibition of MyoD. Gli1 reduced the ability of MyoD to heterodimerize with E12 and bind DNA, providing one mechanism whereby the Gli proteins modulate the activity of MyoD. This novel activity of Gli proteins provides new insights into how Hh signaling modulates terminal differentiation through inhibition of tissue-specific factors such as MyoD. This mechanism may contribute to the broad role of Hh signaling and the Gli proteins in differentiation decisions and cancer formation. PMID:16964293
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Buchdunger, E; Zimmermann, J; Mett, H; Meyer, T; Müller, M; Regenass, U; Lydon, N B
1995-01-01
The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation. Images Fig. 1 Fig. 2 Fig. 3 PMID:7708684
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Su, Lin; Chen, Song-Sen; Yang, Ke-Gong; Liu, Chang-Zheng; Zhang, Yan-Li; Liang, Zhi-Quan
2006-06-01
Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.
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The Hippo signaling pathway provides novel anti-cancer drug targets
Bae, June Sung; Kim, Sun Mi; Lee, Ho
2017-01-01
The Hippo signaling pathway plays a crucial role in cell proliferation, apoptosis, differentiation, and development. Major effectors of the Hippo signaling pathway include the transcriptional co-activators Yes-associated protein 1 (YAP) and WW domain-containing transcription regulator protein 1 (TAZ). The transcriptional activities of YAP and TAZ are affected by interactions with proteins from many diverse signaling pathways as well as responses to the external environment. High YAP and TAZ activity has been observed in many cancer types, and functional dysregulation of Hippo signaling enhances the oncogenic properties of YAP and TAZ and promotes cancer development. Many biological elements, including mechanical strain on the cell, cell polarity/adhesion molecules, other signaling pathways (e.g., G-protein-coupled receptor, epidermal growth factor receptor, Wnt, Notch, and transforming growth factor β/bone morphogenic protein), and cellular metabolic status, can promote oncogenesis through synergistic association with components of the Hippo signaling pathway. Here, we review the signaling networks that interact with the Hippo signaling pathway and discuss the potential of using drugs that inhibit YAP and TAZ activity for cancer therapy. PMID:28035075
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The Hippo signaling pathway provides novel anti-cancer drug targets.
Bae, June Sung; Kim, Sun Mi; Lee, Ho
2017-02-28
The Hippo signaling pathway plays a crucial role in cell proliferation, apoptosis, differentiation, and development. Major effectors of the Hippo signaling pathway include the transcriptional co-activators Yes-associated protein 1 (YAP) and WW domain-containing transcription regulator protein 1 (TAZ). The transcriptional activities of YAP and TAZ are affected by interactions with proteins from many diverse signaling pathways as well as responses to the external environment. High YAP and TAZ activity has been observed in many cancer types, and functional dysregulation of Hippo signaling enhances the oncogenic properties of YAP and TAZ and promotes cancer development. Many biological elements, including mechanical strain on the cell, cell polarity/adhesion molecules, other signaling pathways (e.g., G-protein-coupled receptor, epidermal growth factor receptor, Wnt, Notch, and transforming growth factor β/bone morphogenic protein), and cellular metabolic status, can promote oncogenesis through synergistic association with components of the Hippo signaling pathway. Here, we review the signaling networks that interact with the Hippo signaling pathway and discuss the potential of using drugs that inhibit YAP and TAZ activity for cancer therapy.
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Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R
1996-01-01
The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261
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Mason, Roger M
2013-01-01
Connective tissue growth factor (CTGF, CCN2) is a member of the CCN family of matricellular proteins. It interacts with many other proteins, including plasma membrane proteins, modulating cell function. It is expressed at low levels in normal adult kidney cells but is increased in kidney diseases, playing important roles in inflammation and in the development of glomerular and interstitial fibrosis in chronic disease. This review reports the evidence for its expression in human and animal models of chronic kidney disease and summarizes data showing that anti-CTGF therapy can successfully attenuate fibrotic changes in several such models, suggesting that therapies targeting CTGF and events downstream of it in renal cells may be useful for the treatment of human kidney fibrosis. Connective tissue growth factor stimulates the development of fibrosis in the kidney in many ways including activating cells to increase extracellular matrix synthesis, inducing cell cycle arrest and hypertrophy, and prolonging survival of activated cells. The relationship between CTGF and the pro-fibrotic factor TGFβ is examined and mechanisms by which CTGF promotes signalling by the latter are discussed. No specific cellular receptors for CTGF have been discovered but it interacts with and activates several plasma membrane proteins including low-density lipoprotein receptor-related protein (LRP)-1, LRP-6, tropomyosin-related kinase A, integrins and heparan sulphate proteoglycans. Intracellular signalling and downstream events triggered by such interactions are reviewed. Finally, the relationships between CTGF and several anti-fibrotic factors, such as bone morphogenetic factor-4 (BMP4), BMP7, hepatocyte growth factor, CCN3 and Oncostatin M, are discussed. These may determine whether injured tissue heals or progresses to fibrosis. PMID:23110747
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Kumar, Raj; Singh, Pankaj Kumar; Sharma, Ashok; Prasad, Jagdish; Sagar, Ravinder; Singh, Surender; Arora, Rajesh; Sharma, Rakesh Kumar
2005-08-01
Podophyllum hexandrum Royale (Himalayan mayapple), a high-altitude Himalayan plant, has been shown to provide over 80% whole-body radioprotection in mice. To investigate the radioprotective potential of P. hexandrum at the molecular level, expression patterns of various proteins associated with apoptosis were studied in the spleen of male Swiss albino strain A mice by immunoblotting. Treatment with P. hexandrum [200 mg/kg of body weight; an ethanolic 50% (w/v) extract delivered intraperitoneally] 2 h before irradiation resulted in MAPKAP (mitogen-activated protein kinase-activated protein) kinase-2 activation along with HSF-1 (heat-shock transcription factor-1), leading to up-regulation of HSP-70 (heat-shock protein-70) as compared with sham-irradiated (10 Gy) mice. Strong inhibition of AIF (apoptosis-inducing factor) expression was observed in the mice treated with P. hexandrum 2 h before irradiation as compared with the sham-irradiated group. Inhibition in the translocation of free NF-kappaB (nuclear factor kappaB) from cytoplasm to nucleus was observed upon P. hexandrum pretreatment 2 h before irradiation when compared with radiation-treated mice. P. hexandrum pre-treatment (2 h before irradiation) resulted in inhibition of NF-kappaB translocation, and the expression of tumour suppressor protein p53 was observed to be down-regulated as compared with sham-irradiated control. An increase in the expression of proteins responsible for cell proliferation [Bcl-2 (B-cell chronic lymphocytic lymphoma 2), Ras-GAP (Ras-GTPase-activating protein) and PCNA (proliferating cell nuclear antigen)] was observed in the P. hexandrum-pretreated irradiated mice as compared with sham-irradiated controls. Caspase 3 activation resulted PARP [poly(ADP-ribose) DNA polymerase] cleavage, and DNA degradation was strongly inhibited in the mice treated with P. hexandrm (+/-irradiation) as compared with the mice treated with radiation (+/-heat shock). The present study thus clearly demonstrated that P. hexandrum extract provides protection from gamma-radiation by the modulation of expression of proteins associated with cell death.
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Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease
Naranjo, José R.; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M.; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C.; Arrabal, María D.; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt
2016-01-01
Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648
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Xu, Daichao; Shan, Bing; Lee, Byung-Hoon; Zhu, Kezhou; Zhang, Tao; Sun, Huawang; Liu, Min; Shi, Linyu; Liang, Wei; Qian, Lihui; Xiao, Juan; Wang, Lili; Pan, Lifeng; Finley, Daniel; Yuan, Junying
2015-01-01
Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells. DOI: http://dx.doi.org/10.7554/eLife.10510.001 PMID:26523394
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Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin
2015-03-27
Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Park, Ga Bin; Jeong, Jee-Yeong; Kim, Daejin
2017-01-01
Ampelopsin (Amp) is bioactive natural product and exerts anti-cancer effects against several cancer types. The present study investigated the anti-colon cancer activity of Amp and explored its mechanism of action. The treatment of colon cancer cells with Amp resulted in the dose- and time-dependent induction of apoptosis via the activation of endoplasmic reticulum (ER) stress, 5′ adenosine monophosphate-activated protein kinase (AMPK), and c-Jun N-terminal protein kinase (JNK)/p38 mitogen-activated protein kinases (MAPKs). Salubrinal, an ER stress inhibitor, prevented the upregulation of ER stress-associated proteins, including phosphorylated protein kinase RNA-like ER kinase, phosphorylated eukaryotic translation initiation factor 2α, glucose-regulated protein 78, and CCAAT/enhancer-binding protein homologous protein, as well as suppressing AMPK activation and the MAPK signaling pathway. Knockdown of AMPK by RNA interference failed to block ER stress. Additionally, SP600125 (a JNK inhibitor) and SB203580 (a p38-MAPK inhibitor) effectively inhibited apoptosis and attenuated the expression of X-linked IAP-associated factor 1 (XAF1) and apoptotic Bcl-2 family proteins (BCL2 antagonist/killer 1 and BCL2-associated X protein) in Amp-treated colon cancer cells. Furthermore, reactive oxygen species (ROS)-mediated ER stress/AMPK apoptotic signaling pathway in Amp-treated colon cancer cells were markedly inhibited by treatment with N-acetyl-L-cysteine, a ROS scavenger. These results demonstrate that treatment with Amp induces the apoptotic death of colon cancer cells through ER stress-initiated AMPK/MAPK/XAF1 signaling. These results also provide experimental information for developing Amp as therapeutic drug against colon cancer. PMID:29250183
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Yang, S D; Yu, J S; Lee, T T; Ni, M H; Yang, C C; Ho, Y S; Tsen, T Z
1995-10-01
Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.
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Tissue engineering skeletal muscle for orthopaedic applications
NASA Technical Reports Server (NTRS)
Payumo, Francis C.; Kim, Hyun D.; Sherling, Michael A.; Smith, Lee P.; Powell, Courtney; Wang, Xiao; Keeping, Hugh S.; Valentini, Robert F.; Vandenburgh, Herman H.
2002-01-01
With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.
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Jana, Arundhati; Modi, Khushbu K; Roy, Avik; Anderson, John A; van Breemen, Richard B; Pahan, Kalipada
2013-06-01
This study underlines the importance of cinnamon, a widely-used food spice and flavoring material, and its metabolite sodium benzoate (NaB), a widely-used food preservative and a FDA-approved drug against urea cycle disorders in humans, in increasing the levels of neurotrophic factors [e.g., brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3)] in the CNS. NaB, but not sodium formate (NaFO), dose-dependently induced the expression of BDNF and NT-3 in primary human neurons and astrocytes. Interestingly, oral administration of ground cinnamon increased the level of NaB in serum and brain and upregulated the levels of these neurotrophic factors in vivo in mouse CNS. Accordingly, oral feeding of NaB, but not NaFO, also increased the level of these neurotrophic factors in vivo in the CNS of mice. NaB induced the activation of protein kinase A (PKA), but not protein kinase C (PKC), and H-89, an inhibitor of PKA, abrogated NaB-induced increase in neurotrophic factors. Furthermore, activation of cAMP response element binding (CREB) protein, but not NF-κB, by NaB, abrogation of NaB-induced expression of neurotrophic factors by siRNA knockdown of CREB and the recruitment of CREB and CREB-binding protein to the BDNF promoter by NaB suggest that NaB exerts its neurotrophic effect through the activation of CREB. Accordingly, cinnamon feeding also increased the activity of PKA and the level of phospho-CREB in vivo in the CNS. These results highlight a novel neutrophic property of cinnamon and its metabolite NaB via PKA - CREB pathway, which may be of benefit for various neurodegenerative disorders.
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Akt-Dependent Cytokine Production in Mast Cells
Kitaura, Jiro; Asai, Koichi; Maeda-Yamamoto, Mari; Kawakami, Yuko; Kikkawa, Ushio; Kawakami, Toshiaki
2000-01-01
Cross-linking of FcεRI induces the activation of three protein tyrosine kinases, Lyn, Syk, and Bruton's tyrosine kinase (Btk), leading to the secretion of a panel of proinflammatory mediators from mast cells. This study showed phosphorylation at Ser-473 and enzymatic activation of Akt/protein kinase B, the crucial survival kinase, upon FcεRI stimulation in mouse mast cells. Phosphorylation of Akt is regulated positively by Btk and Syk and negatively by Lyn. Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-α promoters. Transcription from the nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1) sites within these promoters is under the control of Akt activity. Accordingly, the signaling pathway involving IκB-α, a cytoplasmic protein that binds NF-κB and inhibits its nuclear translocation, appears to be regulated by Akt in mast cells. Catalytic activity of glycogen synthase kinase (GSK)-3β, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt. Importantly, Akt regulates the production and secretion of IL-2 and TNF-α in FcεRI-stimulated mast cells. Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-κB, NF-AT, and AP-1 that contributes to the production of cytokines. PMID:10974038
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Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins
Zhao, Baoyu; Shu, Chang; Gao, Xinsheng; ...
2016-06-02
Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here in this paper, we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF bindsmore » to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.« less
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Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhao, Baoyu; Shu, Chang; Gao, Xinsheng
Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here in this paper, we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF bindsmore » to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.« less
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Genetics Home Reference: aniridia
... PAX6 protein attaches (binds) to specific regions of DNA and regulates the activity of other genes. On the basis of this role, the PAX6 protein is called a transcription factor. Following birth, the PAX6 protein regulates several ...
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Karimpour, Shervin; Lou, Junyang; Lin, Lilie L; Rene, Luis M; Lagunas, Lucio; Ma, Xinrong; Karra, Sreenivasu; Bradbury, C Matthew; Markovina, Stephanie; Goswami, Prabhat C; Spitz, Douglas R; Hirota, Kiichi; Kalvakolanu, Dhananjaya V; Yodoi, Junji; Gius, David
2002-09-12
A recently identified class of signaling factors uses critical cysteine motif(s) that act as redox-sensitive 'sulfhydryl switches' to reversibly modulate specific signal transduction cascades regulating downstream proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and transcription factors such as the AP-1 complex both contain redox-sensitive cysteine motifs that regulate activity in response to oxidative stress. The mammalian thioredoxin reductase-1 (TR) is an oxidoreductase selenocysteine-containing flavoprotein that also appears to regulate multiple downstream intracellular redox-sensitive proteins. Since ionizing radiation (IR) induces oxidative stress as well as increases AP-1 DNA-binding activity via the activation of Ref-1, the potential roles of TR and thioredoxin (TRX) in the regulation of AP-1 activity in response to IR were investigated. Permanently transfected cell lines that overexpress wild type TR demonstrated constitutive increases in AP-1 DNA-binding activity as well as AP-1-dependent reporter gene expression, relative to vector control cells. In contrast, permanently transfected cell lines expressing a TR gene with the active site cysteine motif deleted were unable to induce AP-1 activity or reporter gene expression in response to IR. Transient genetic overexpression of either the TR wild type or dominant-negative genes demonstrated similar results using a transient assay system. One mechanism through which TR regulates AP-1 activity appears to involve TRX sub-cellular localization, with no change in the total TRX content of the cell. These results identify a novel function of the TR enzyme as a signaling factor in the regulation of AP-1 activity via a cysteine motif located in the protein.
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Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.
Disatnik, M H; Hernandez-Sotomayor, S M; Jones, G; Carpenter, G; Mochly-Rosen, D
1994-01-18
Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.
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Graness, A; Hanke, S; Boehmer, F D; Presek, P; Liebmann, C
2000-01-01
Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR. PMID:10749673
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Insulin/IGF-1 signaling mutants reprogram ER stress response regulators to promote longevity.
Henis-Korenblit, Sivan; Zhang, Peichuan; Hansen, Malene; McCormick, Mark; Lee, Seung-Jae; Cary, Michael; Kenyon, Cynthia
2010-05-25
When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response is activated. This ER stress response restores ER homeostasis by coordinating processes that decrease translation, degrade misfolded proteins, and increase the levels of ER-resident chaperones. Ribonuclease inositol-requiring protein-1 (IRE-1), an endoribonuclease that mediates unconventional splicing, and its target, the XBP-1 transcription factor, are key mediators of the unfolded protein response. In this study, we show that in Caenorhabditis elegans insulin/IGF-1 pathway mutants, IRE-1 and XBP-1 promote lifespan extension and enhance resistance to ER stress. We show that these effects are not achieved simply by increasing the level of spliced xbp-1 mRNA and expression of XBP-1's normal target genes. Instead, in insulin/IGF-1 pathway mutants, XBP-1 collaborates with DAF-16, a FOXO-transcription factor that is activated in these mutants, to enhance ER stress resistance and to activate new genes that promote longevity.
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Small-Molecule Inhibitors of the SOX18 Transcription Factor.
Fontaine, Frank; Overman, Jeroen; Moustaqil, Mehdi; Mamidyala, Sreeman; Salim, Angela; Narasimhan, Kamesh; Prokoph, Nina; Robertson, Avril A B; Lua, Linda; Alexandrov, Kirill; Koopman, Peter; Capon, Robert J; Sierecki, Emma; Gambin, Yann; Jauch, Ralf; Cooper, Matthew A; Zuegg, Johannes; Francois, Mathias
2017-03-16
Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Showalter, Aaron D; Smith, Timothy P L; Bennett, Gary L; Sloop, Kyle W; Whitsett, Julie A; Rhodes, Simon J
2002-05-29
The Prophet of Pit-1 (PROP1) gene encodes a paired class homeodomain transcription factor that is exclusively expressed in the developing mammalian pituitary gland. PROP1 function is essential for anterior pituitary organogenesis, and heritable mutations in the gene are associated with combined pituitary hormone deficiency in human patients and animals. By cloning the bovine PROP1 gene and by comparative analysis, we demonstrate that the homeodomains and carboxyl termini of mammalian PROP1 proteins are highly conserved while the amino termini are diverged. Whereas the carboxyl termini of the human and bovine PROP1 proteins contain potent transcriptional activation domains, the amino termini and homeodomains have repressive activities. The bovine PROP1 gene has four exons and three introns and maps to a region of chromosome seven carrying a quantitative trait locus affecting ovulation rate. Two alleles of the bovine gene were found that encode distinct protein products with different DNA binding and transcriptional activities. These experiments demonstrate that mammalian PROP1 genes encode proteins with complex regulatory capacities and that modest changes in protein sequence can significantly alter the activity of this pituitary developmental transcription factor.
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Zamanian, M; La Thangue, N B
1992-01-01
The retinoblastoma (Rb) gene product forms a complex with the cellular transcription factor DRTF1, a property assumed to be important for mediating negative growth control because certain viral oncogenes, such as adenovirus E1a, prevent this interaction and mutant Rb alleles, which have lost the capacity to regulate growth, encode proteins that fail to associate with DRTF1. In this study, we show that the wild-type Rb protein can specifically repress transcription from promoters driven by DRTF1 whereas a naturally occurring mutant Rb protein cannot. Furthermore, Rb-mediated transcriptional repression can be overridden by adenovirus E1a; this requires regions in E1a necessary for cellular transformation. The Rb protein therefore acts in trans to repress the transcriptional activity of DRTF1 whereas adenovirus E1a prevents this interaction and thus maintains DRTF1 in a constitutively active state. The Rb protein and adenovirus E1a therefore have opposite effects on the activity of a common molecular target. Transcriptional repression mediated by the Rb protein and inactivation of repression by the E1a protein are likely to play an important role in mediating their biological effects. Images PMID:1385776
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Nojima, Junzo; Kuratsune, Hirohiko; Suehisa, Etsuji; Kawasaki, Tomio; Machii, Takashi; Kitani, Teruo; Iwatani, Yoshinori; Kanakura, Yuzuru
2002-11-01
Anti-phospholipid (aPL) antibodies (Abs) are well known to be associated with thromboembolic events in patients with systemic lupus erythematosus (SLE). However, the clinical relevance of a PL Abs in patients without SLE (non-SLE) who have venous thromboembolism remains unclear. We evaluated 143 non-SLE patients with a first episode of clinically suspected deep vein thrombosis (DVT) by using objective tests for diagnosing DVT and laboratory tests including the activated protein C resistance (APC-R) test, the factor V Leiden test, and various aPL Abs. The prevalence of acquired APC-R, in which case there was no factor V Leiden mutation, was significantly higher in patients with DVT (15/58 cases, 25.9%, p < 0.0001) than in those without DVT (3/80 cases, 3.7%), and confirmed that acquired APC-R was a strong risk factor for DVT (odds ratio [OR], 8.95; 95% confidence intervals [CI], 2.45-32.7; p < 0.001). Multivariate logistic analysis revealed that the presence of LA, aCL, anti-beta2-glycoprotein I, anti-prothrombin and anti-protein C Abs was not reliable as a risk factor for DVT in non-SLE patients, and that the presence of anti-protein S Abs was the most significant risk factor for DVT (OR, 5.88; 95% CI, 1.96-17.7; p < 0.002). Furthermore, the presence of anti-protein S Abs was strongly associated with acquired APC-R (OR, 57.8; 95% CI, 8.53-391; p < 0.0001). These results suggest that acquired APC-R may reflect functional interference by anti-protein S Abs of the protein C pathway, which action may represent an important mechanism for the development DVT in non-SLE patients.
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Laev, Sergey S; Salakhutdinov, Nariman F; Lavrik, Olga I
2017-05-01
Human apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein which is essential in the base excision repair (BER) pathway of DNA lesions caused by oxidation and alkylation. This protein hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety or activates the DNA-binding activity of certain transcription factors through its redox function. Studies have indicated a role for APE1/Ref-1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs. Thus, this protein has potential as a target in cancer treatment. As a result, major efforts have been directed to identify small molecule inhibitors against APE1/Ref-1 activities. These agents have the potential to become anticancer drugs. The aim of this review is to present recent progress in studies of all published small molecule APE1/Ref-1 inhibitors. The structures and activities of APE1/Ref-1 inhibitors, that target both DNA repair and redox activities, are presented and discussed. To date, there is an urgent need for further development of the design and synthesis of APE1/Ref-1 inhibitors due to high importance of this protein target. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Yang, Liu; Sha, Haibo; Davisson, Robin L; Qi, Ling
2013-05-10
The cross-talk between UPR activation and metabolic stress remains largely unclear. Phenformin treatment activates the IRE1α and PERK pathways in an AMPK-dependent manner. AMPK is required for phenformin-mediated IRE1α and PERK activation. Our findings demonstrate the cross-talk between UPR and metabolic signals. Activation of the unfolded protein response (UPR) is associated with the disruption of endoplasmic reticulum (ER) homeostasis and has been implicated in the pathogenesis of many human metabolic diseases, including obesity and type 2 diabetes. However, the nature of the signals activating UPR under these conditions remains largely unknown. Using a method that we recently optimized to directly measure UPR sensor activation, we screened the effect of various metabolic drugs on UPR activation and show that the anti-diabetic drug phenformin activates UPR sensors IRE1α and pancreatic endoplasmic reticulum kinase (PERK) in both an ER-dependent and ER-independent manner. Mechanistically, AMP-activated protein kinase (AMPK) activation is required but not sufficient to initiate phenformin-mediated IRE1α and PERK activation, suggesting the involvement of additional factor(s). Interestingly, activation of the IRE1α (but not PERK) pathway is partially responsible for the cytotoxic effect of phenformin. Together, our data show the existence of a non-canonical UPR whose activation requires the cytosolic kinase AMPK, adding another layer of complexity to UPR activation upon metabolic stress.
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YAN, FANG; CAO, HANWEI; COVER, TIMOTHY L.; WHITEHEAD, ROBERT; WASHINGTON, M. KAY; POLK, D. BRENT
2011-01-01
Background & Aims Increased inflammatory cytokine levels and intestinal epithelial cell apoptosis leading to disruption of epithelial integrity are major pathologic factors in inflammatory bowel diseases. The probiotic bacterium Lactobacillus rhamnosus GG (LGG) and factors recovered from LGG broth culture supernatant (LGG-s) prevent cytokine-induced apoptosis in human and mouse intestinal epithelial cells by regulating signaling pathways. Here, we purify and characterize 2 secreted LGG proteins that regulate intestinal epithelial cell antiapoptotic and proliferation responses. Methods LGG proteins were purified from LGG-s, analyzed, and used to generate polyclonal antibodies for immunodepletion of respective proteins from LGG-conditioned cell culture media (CM). Mouse colon epithelial cells and cultured colon explants were treated with purified proteins in the absence or presence of tumor necrosis factor (TNF). Akt activation, proliferation, tissue injury, apoptosis, and caspase-3 activation were determined. Results We purified 2 novel proteins, p75 (75 kilodaltons) and p40 (40 kilodaltons), from LGG-s. Each of these purified protein preparations activated Akt, inhibited cytokine-induced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. TNF-induced colon epithelial damage was significantly reduced by p75 and p40. Immunodepletion of p75 and p40 from LGG-CM reversed LGG-CM activation of Akt and its inhibitory effects on cytokine-induced apoptosis and loss of intestinal epithelial cells. Conclusions p75 and p40 are the first probiotic bacterial proteins demonstrated to promote intestinal epithelial homeostasis through specific signaling pathways. These findings suggest that probiotic bacterial components may be useful for preventing cytokine-mediated gastrointestinal diseases. PMID:17258729
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Functional characterization of the turkey macrophage migration inhibitory factor
USDA-ARS?s Scientific Manuscript database
Macrophage migration inhibitory factor (MIF) is a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. The aim of this study was to clone the turkey MIF (TkMIF) gene, express the active protein, and characte...
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Kpna7 interacts with egg-specific nuclear factors in rainbow trout (Oncorhynchus mykiss)
USDA-ARS?s Scientific Manuscript database
Nuclear proteins are required for initiation of transcription in early embryos before embryonic genome activation. The regulation of transportation of nuclear proteins is mediated by transport factors known as importins (karyopherins). Kpna7 is a newly discovered member of the importin a family, whi...
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Acetyllysine-binding and function of bromodomain-containing proteins in chromatin.
Dyson, M H; Rose, S; Mahadevan, L C
2001-08-01
Acetylated histones are generally associated with active chromatin. The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin. Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes. Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin. Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function.
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Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation.
Nilsson, Kersti; Wu, Chengjun; Schwartz, Stefan
2018-06-12
Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.
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DNA-binding activity of TNF-{alpha} inducing protein from Helicobacter pylori
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kuzuhara, T.; Suganuma, M.; Oka, K.
2007-11-03
Tumor necrosis factor-{alpha} (TNF-{alpha}) inducing protein (Tip{alpha}) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-{alpha} and chemokine genes and activation of nuclear factor-{kappa}B. Since Tip{alpha} enters gastric cancer cells, the Tip{alpha} binding molecules in the cells should be investigated. The direct DNA-binding activity of Tip{alpha} was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tip{alpha} and DNA, revealed that the affinity of Tip{alpha} for (dGdC)10 is 2400 times stronger than that of del-Tip{alpha}, an inactive Tip{alpha}. This suggestsmore » a strong correlation between DNA-binding activity and carcinogenic activity of Tip{alpha}. And the DNA-binding activity of Tip{alpha} was first demonstrated with a molecule secreted from H. pylori.« less
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Xiong, Ying; Zhang, Jie; Liu, Man; An, Mingwei; Lei, Ling; Guo, Wuhua
2014-09-01
Current treatment modalities for various types of hepatic cancer, which has an increasing incidence rate, are inadequate and novel therapies are required. Therefore, identifying targets for liver cancer is becoming increasingly valuable to develop novel methods for therapy. The aim of the present study was to examine the growth activation mechanism of the leptin protein in the liver cancer cell line HepG2. The effects of the leptin protein on cell death were investigated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide analysis. DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis were also performed to detect cell apoptosis. The expression of leptin and three endoplasmic reticulum (ER) stress unfolded protein response (UPR) proteins, including activating transcription factor 6, phosphorylated‑PKR‑like ER kinase (p‑PERK) and inositol requiring protein 1, were investigated for the examination of ER stress. The mRNA UPR proteins were also detected by reverse transcription polymerase chain reaction. The apoptosis‑associated caspase 12 and C/EBP homologous protein (CHOP) was detected by western blot analysis. The expression of or incubation with the leptin protein was able to activate cell growth and inhibit cell death and apoptosis. In cells that expressed leptin or were incubated with leptin protein (pep-LPT), cisplatin‑induced ER stress‑associated mRNA transcription and protein activation were inhibited. Levels of the ER stress UPR pathway protein, PERK, increased significantly in leptin‑silenced cells when treated with cisplatin as compared with those in the leptin‑expressing or pep-LPT cells. Furthermore, caspase 12 activation was inhibited in ex‑LPT, pep‑LPT and HepG2 cells. In conclusion, human leptin protein is involved in promoting the proliferation of HepG2 cells through inhibiting the ER stress‑associated apoptotic pathway. The PERK UPR pathway and the apoptotic factor caspase 12 were found to be involved in the inhibition of apoptosis and enhancement of proliferation.
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Huh, Jeong-Eun; Lee, Wong In; Kang, Jung Won; Nam, Dongwoo; Choi, Do-Young; Park, Dong-Suk; Lee, Sang Hoon; Lee, Jae-Dong
2014-11-26
Formononetin (1), a plant-derived phytoestrogen, possesses bone protective properties. To address the potential therapeutic efficacy and mechanism of action of 1, we investigated its antiosteoclastogenic activity and its effect on nuclear factor-kappaB ligand (RANKL)-induced bone-marrow-derived macrophages (BMMs). Compound 1 markedly inhibited RANKL-induced osteoclast differentiation in the absence of cytotoxicity, by regulating the expression of osteoprotegerin (OPG) and RANKL in BMMs and in cocultured osteoblasts. Compound 1 significantly inhibited RANKL-induced tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1α (MIP-1α) in a concentration-dependent manner. These effects were accompanied by a decrease in RANKL-induced activation of the NF-κB p65 subunit, degradation of inhibitor κBα (IκBα), induction of NF-κB, and phosphorylation of AKT, extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). NF-κB siRNA suppressed AKT, ERK, JNK, and p38 MAPK phosphorylation. Furthermore, 1 significantly suppressed c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), key transcription factors during osteoclastogenesis. SP600125, a specific inhibitor of JNK, reduced RANKL-induced expression of phospho-c-Jun, c-Fos, and NFATc1 and inhibited osteoclast formation. These results suggested that 1 acted as an antiresorption agent by blocking osteoclast activation.
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Hamel, Louis-Philippe; Benchabane, Meriem; Nicole, Marie-Claude; Major, Ian T.; Morency, Marie-Josée; Pelletier, Gervais; Beaudoin, Nathalie; Sheen, Jen; Séguin, Armand
2011-01-01
Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors. PMID:21873571
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Gondard, Elise; Chau, Hien N; Mann, Amandeep; Tierney, Travis S; Hamani, Clement; Kalia, Suneil K; Lozano, Andres M
2015-01-01
The forniceal area is currently being evaluated as a target for deep brain stimulation (DBS) to improve cognitive function in patients with Alzheimer's disease. The molecular changes at downstream targets within the stimulated circuit are unknown. To analyze the modulation of hippocampal protein expression following 1 h of fornix DBS in the rat. Animals underwent bilateral forniceal DBS for 1 h and sacrificed at different time-points after the initiation of the stimulation (1 h, 2.5 h, 5 h, 25 h). Bilateral hippocampi were isolated for western blot analyses. Forniceal DBS led to a dramatic elevation of cFos post-stimulation, suggesting that forniceal DBS activates the hippocampus. There was also a significant increase in candidate proteins including several trophic factors, such as brain derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) but not glial cell-derived neurotrophic factor (GDNF). There was in addition, increased expression of the synaptic markers growth associated protein 43 (GAP-43), synaptophysin and α-synuclein. No changes were observed at the studied time-points in Alzheimer's-related proteins including amyloid precursor protein (APP), tau, phosphorylated tau (ptau), or selected chaperone proteins (HSP40, HSP70 and CHIP). Forniceal DBS triggers hippocampal activity and rapidly modulate the expression of neurotrophic factors and markers of synaptic plasticity known to play key roles in memory processing. The clinical effects of DBS of the fornix may, in part, be mediated by producing changes in the expression of these proteins. Copyright © 2015 Elsevier Inc. All rights reserved.
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Luo, Hongmin; Sawashita, Jinko; Tian, Geng; Liu, Yingye; Li, Lin; Ding, Xin; Xu, Zhe; Yang, Mu; Miyahara, Hiroki; Mori, Masayuki; Qian, Jinze; Wang, Yaoyong; Higuchi, Keiichi
2015-03-01
Mouse senile amyloidosis is a disorder in which apolipoprotein A-II deposits extracellularly in many organs as amyloid fibrils (AApoAII). In this study, we intravenously injected 1 μg of isolated AApoAII fibrils into R1.P1-Apoa2(c) mice, to induce AApoAII amyloidosis. We observed that the unfolded protein response was induced by deposition of AApoAII amyloid. We found that the mRNA and the protein expression levels of heat shock protein A5 (HSPA5; also known as glucose-regulated protein 78) were increased in the liver with AApoAII amyloid deposits. Immunohistochemistry showed that HSPA5 was only detected in hepatocytes close to AApoAII amyloid deposits. Furthermore, gene transcription of several endoplasmic reticulum (ER) stress-related proteins increased, including eukaryotic translation initiation factor 2 alpha kinase 3 (Eif2ak3), activating transcription factor 6 (Atf6), activating transcription factor 4 (Atf4), X-box-binding protein 1 splicing (Xbp1s), DNA-damage inducible transcript 3 (Ddit3), and autophagy protein 5 (Atg5). Moreover, apoptosis-positive cells were increased in the liver. Similar results were seen in the kidney but not in the heart. Our study indicates that ER stress responses differed among tissues with extracellular AApoAII amyloid fibril deposition. Although upregulated HSPA5 and the activated unfolded protein response might have roles in protecting tissues against aggregated extracellular AApoAII amyloid deposition, prolonged ER stress induced apoptosis in the liver and the kidney.
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Identification of a G protein coupled receptor induced in activated T cells.
Kaplan, M H; Smith, D I; Sundick, R S
1993-07-15
Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.
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Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R.; Hellmann, Hanjo
2013-01-01
Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that CULLIN3-based E3 ligases have the potential to interact with a broad range of ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 (AP2) transcription factors, mediated by MATH-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members. PMID:23792371
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Rushton, Paul J; Somssich, Imre E; Ringler, Patricia; Shen, Qingxi J
2010-05-01
WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy. 2010 Elsevier Ltd. All rights reserved.
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Sooranna, S R; Engineer, N; Liang, Z; Bennett, P R; Johnson, M R
2007-07-01
IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.
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Metformin targets multiple signaling pathways in cancer.
Lei, Yong; Yi, Yanhua; Liu, Yang; Liu, Xia; Keller, Evan T; Qian, Chao-Nan; Zhang, Jian; Lu, Yi
2017-01-26
Metformin, an inexpensive and well-tolerated oral agent commonly used in the first-line treatment of type 2 diabetes, has become the focus of intense research as a candidate anticancer agent. Here, we discuss the potential of metformin in cancer therapeutics, particularly its functions in multiple signaling pathways, including AMP-activated protein kinase, mammalian target of rapamycin, insulin-like growth factor, c-Jun N-terminal kinase/mitogen-activated protein kinase (p38 MAPK), human epidermal growth factor receptor-2, and nuclear factor kappaB pathways. In addition, cutting-edge targeting of cancer stem cells by metformin is summarized.
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Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity
SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.
2016-01-01
SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280
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Oh, Chorong; Jeon, Byeong Hwan; Reid Storm, Shaun Nicholas; Jho, Sunkug; No, Jae-Kyung
2017-01-01
The aim of this study was to evaluate the effects of the types and levels of physical activity in conjunction with protein intake and vitamin D on sarcopenia and obesity status in an elderly population. Study participants (N = 4452) were ages ≥60 y and included 1929 men and 2523 women who completed a body composition analysis with a dual energy x-ray absorptiometry and provided health and dietary data. Higher appendicular skeletal muscle mass/weight was observed in the non-obese group, although obese participants had greater weights. The non-obese sarcopenia subgroup showed health problems related to insulin resistance and metabolic-related factors compared with the nonsarcopenic group. The total metabolic equivalent was significantly different in both obese categories, regardless of sarcopenic status. The prevalence of obesity, sarcopenia, and sarcopenic obesity relatively increased with a diet deficient of protein intake and vitamin D. These data suggest that sarcopenia had a significant association with metabolic-related factors; physical activity, especially vigorous activity; and protein intake and vitamin D levels in a non-obese elderly population. Therefore, maintaining healthy body weight by means of resistance exercise and enhanced protein intake and vitamin D may help offset sarcopenia in this age group. Copyright © 2016 Elsevier Inc. All rights reserved.
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Sadi, Gökhan; Bozan, Davut; Yildiz, Huseyin Bekir
2014-08-01
Resveratrol is a strong antioxidant that exhibits blood glucose-lowering effects, which might contribute to its usefulness in preventing complications associated with diabetes. The present study aimed to investigate resveratrol effects on catalase (CAT) and glutathione peroxidase (GPx) gene and protein expression, their phosphorylation states and activities in rat liver of STZ-induced diabetes. Diabetes increased the levels of total protein phosphorylation and p-CAT, while mRNA expression, protein levels, and activity were reduced. Although diabetes induced transcriptional repression over GPx, it did not affect the protein levels and activity. When resveratrol was administered to diabetic rats, an increase in activity was associated with an increase in p-GPx levels. Decrease in Sirtuin1 (SIRT1) and nuclear factor erythroid 2-related factor (Nrf2) and increase in nuclear factor kappa B (NFκB) gene expression in diabetes were associated with a decrease in CAT and GPx mRNA expression. A possible compensatory mechanism for reduced gene expression of antioxidant enzymes is proved to be nuclear translocation of redox-sensitive Nrf2 and NFκB in diabetes which is confirmed by the increase in nuclear and decrease in cytoplasmic protein levels of Nrf2 and NFκB. Taken together, these findings revealed that an increase in the oxidized state in diabetes intricately modified the cellular phosphorylation status and regulation of antioxidant enzymes. Gene regulation of antioxidant enzymes was accompanied by nuclear translocation of Nrf2 and NFκB. Resveratrol administration also activated a coordinated cytoprotective response against diabetes-induced changes in liver tissues.
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del Valle, Luis J
2005-06-01
Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg-jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø(o) (sperm-specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca(2+), pH, and this activity could be a serine-proteinase. Thermal denaturalization of the oocyte extracts (80 degrees C, 10-15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø(o)) will be a first condition in the process of sperm chromatin remodeling.
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Vergis, James M.; Wiener, Michael C.
2011-01-01
Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (Enterokinase, Factor Xa, Human Rhinovirus 3C Protease, SUMOstar, Tobacco Etch Virus Protease, and Thrombin) by use of a panel of ninety-four individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and Factor Xa were only affected by a small number of detergents, making them good choices as well. PMID:21539919
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Brain-derived neurotrophic factor and its receptors in Bergmann glia cells.
Poblete-Naredo, Irais; Guillem, Alain M; Juárez, Claudia; Zepeda, Rossana C; Ramírez, Leticia; Caba, Mario; Hernández-Kelly, Luisa C; Aguilera, José; López-Bayghen, Esther; Ortega, Arturo
2011-12-01
Brain-derived neurotrophic factor is an abundant and widely distributed neurotrophin expressed in the Central Nervous System. It is critically involved in neuronal differentiation and survival. The expression of brain-derived neurotrophic factor and that of its catalytic active cognate receptor (TrkB) has been extensively studied in neuronal cells but their expression and function in glial cells is still controversial. Despite of this fact, brain-derived neurotrophic factor is released from astrocytes upon glutamate stimulation. A suitable model to study glia/neuronal interactions, in the context of glutamatergic synapses, is the well-characterized culture of chick cerebellar Bergmann glia cells. Using, this system, we show here that BDNF and its functional receptor are present in Bergmann glia and that BDNF stimulation is linked to the activation of the phosphatidyl-inositol 3 kinase/protein kinase C/mitogen-activated protein kinase/Activator Protein-1 signaling pathway. Accordingly, reverse transcription-polymerase chain reaction (RT-PCR) experiments predicted the expression of full-length and truncated TrkB isoforms. Our results suggest that Bergmann glia cells are able to express and respond to BDNF stimulation favoring the notion of their pivotal role in neuroprotection. Copyright © 2011 Elsevier B.V. All rights reserved.
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Generation of Viable Cell and Biomaterial Patterns by Laser Transfer
NASA Astrophysics Data System (ADS)
Ringeisen, Bradley
2001-03-01
In order to fabricate and interface biological systems for next generation applications such as biosensors, protein recognition microarrays, and engineered tissues, it is imperative to have a method of accurately and rapidly depositing different active biomaterials in patterns or layered structures. Ideally, the biomaterial structures would also be compatible with many different substrates including technologically relevant platforms such as electronic circuits or various detection devices. We have developed a novel laser-based technique, termed matrix assisted pulsed laser evaporation direct write (MAPLE DW), that is able to direct write patterns and three-dimensional structures of numerous biologically active species ranging from proteins and antibodies to living cells. Specifically, we have shown that MAPLE DW is capable of forming mesoscopic patterns of living prokaryotic cells (E. coli bacteria), living mammalian cells (Chinese hamster ovaries), active proteins (biotinylated bovine serum albumin, horse radish peroxidase), and antibodies specific to a variety of classes of cancer related proteins including intracellular and extracellular matrix proteins, signaling proteins, cell cycle proteins, growth factors, and growth factor receptors. In addition, patterns of viable cells and active biomolecules were deposited on different substrates including metals, semiconductors, nutrient agar, and functionalized glass slides. We will present an explanation of the laser-based transfer mechanism as well as results from our recent efforts to fabricate protein recognition microarrays and tissue-based microfluidic networks.
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Dälken, Benjamin; Jabulowsky, Robert A.; Oberoi, Pranav; Benhar, Itai; Wels, Winfried S.
2010-01-01
Background The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins. PMID:21203542
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Genetics Home Reference: nail-patella syndrome
... protein that attaches (binds) to specific regions of DNA and regulates the activity of other genes. On the basis of this role, the LMX1B protein is called a transcription factor. The LMX1B protein appears to be particularly ...
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A role for NRAGE in NF-κB activation through the non-canonical BMP pathway
2010-01-01
Background Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-κB has yet to be explored. Results Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -α/β and subsequent transcriptional activation of the p65 subunit of NF-κB. Ablation of endogenous NRAGE by siRNA inhibited NF-κB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-κB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor. Conclusion Modulation of NRAGE expression revealed novel roles in regulating NF-κB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway. PMID:20100315
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Diling, Chen; Chaoqun, Zheng; Jian, Yang; Jian, Li; Jiyan, Su; Yizhen, Xie; Guoxiao, Lai
2017-01-01
A single-band protein (HEP3) was isolated from Hericium erinaceus using a chemical separation combined with pharmacodynamic evaluation methods. This protein exhibited immunomodulatory activity in lipopolysaccharide-activated RAW 264.7 macrophages by decreasing the overproduction of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and downregulating the expression of inducible nitric oxide synthase and nuclear factor-κB p65. Further researches revealed that HEP3 could improve the immune system via regulating the composition and metabolism of gut microbiota to activate the proliferation and differentiation of T cells, stimulate the intestinal antigen-presenting cells in high-dose cyclophosphamide-induced immunotoxicity in mice, and play a prebiotic role in the case of excessive antibiotics in inflammatory bowel disease model mice. Aided experiments also showed that HEP3 could be used as an antitumor immune inhibitor in tumor-burdened mice. The results of the present study suggested that fungal protein from H. erinaceus could be used as a drug or functional food ingredient for immunotherapy because of its immunomodulatory activities.
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Diling, Chen; Chaoqun, Zheng; Jian, Yang; Jian, Li; Jiyan, Su; Yizhen, Xie; Guoxiao, Lai
2017-01-01
A single-band protein (HEP3) was isolated from Hericium erinaceus using a chemical separation combined with pharmacodynamic evaluation methods. This protein exhibited immunomodulatory activity in lipopolysaccharide-activated RAW 264.7 macrophages by decreasing the overproduction of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and downregulating the expression of inducible nitric oxide synthase and nuclear factor-κB p65. Further researches revealed that HEP3 could improve the immune system via regulating the composition and metabolism of gut microbiota to activate the proliferation and differentiation of T cells, stimulate the intestinal antigen-presenting cells in high-dose cyclophosphamide-induced immunotoxicity in mice, and play a prebiotic role in the case of excessive antibiotics in inflammatory bowel disease model mice. Aided experiments also showed that HEP3 could be used as an antitumor immune inhibitor in tumor-burdened mice. The results of the present study suggested that fungal protein from H. erinaceus could be used as a drug or functional food ingredient for immunotherapy because of its immunomodulatory activities. PMID:28713364
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Olsen, J; Lefebvre, O; Fritsch, C; Troelsen, J T; Orian-Rousseau, V; Kedinger, M; Simon-Assmann, P
2000-01-01
Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF. PMID:10749670
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Iyer, Janaki Krishnamurthy; Shih, Norrapat; Majumder, Munmi; Mattaparthi, Venkata Satish Kumar; Mukhopadhyay, Rupak; Doley, Robin
2016-01-01
In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity. PMID:27089306
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GIV/Girdin activates Gαi and inhibits Gαs via the same motif
Gupta, Vijay; Bhandari, Deepali; Leyme, Anthony; Aznar, Nicolas; Midde, Krishna K.; Lo, I-Chung; Ear, Jason; Niesman, Ingrid; López-Sánchez, Inmaculada; Blanco-Canosa, Juan Bautista; von Zastrow, Mark; Garcia-Marcos, Mikel; Farquhar, Marilyn G.; Ghosh, Pradipta
2016-01-01
We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK–ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV–Gαi complexes and activates GIV’s GEF function; then a second phosphomodification terminates GIV’s GEF function, triggers the assembly of GIV–Gαs complexes, and activates GIV’s GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK–ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses. PMID:27621449
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Suryawan, Agus; Jeyapalan, Asumthia S; Orellana, Renan A; Wilson, Fiona A; Nguyen, Hanh V; Davis, Teresa A
2008-10-01
Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.
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Suryawan, Agus; Jeyapalan, Asumthia S.; Orellana, Renan A.; Wilson, Fiona A.; Nguyen, Hanh V.; Davis, Teresa A.
2008-01-01
Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E·eIF4G complex and increased eIF4E·4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein β-subunit-like protein (GβL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors. PMID:18682538
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Sankhala, Rajeshwer S; Damai, Rajani S; Swamy, Musti J
2011-03-08
The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109.
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Prokova, Vassiliki; Mavridou, Sofia; Papakosta, Paraskevi; Kardassis, Dimitris
2005-01-01
Transforming growth factor beta (TGFbeta) regulates transcriptional responses via activation of cytoplasmic effector proteins termed Smads. Following their phosphorylation by the type I TGFbeta receptor, Smads form oligomers and translocate to the nucleus where they activate the transcription of TGFbeta target genes in cooperation with nuclear cofactors and coactivators. In the present study, we have undertaken a deletion analysis of human Smad3 protein in order to characterize domains that are essential for transcriptional activation in mammalian cells. With this analysis, we showed that Smad3 contains two domains with transcriptional activation function: the MH2 domain and a second middle domain that includes the linker region and the first two beta strands of the MH2 domain. Using a protein-protein interaction assay based on biotinylation in vivo, we were able to show that a Smad3 protein bearing an internal deletion in the middle transactivation domain is characterized by normal oligomerization and receptor activation properties. However, this mutant has reduced transactivation capacity on synthetic or natural promoters and is unable to interact physically and functionally with the histone acetyltransferase p/CAF. The loss of interaction with p/CAF or other coactivators could account, at least in part, for the reduced transactivation capacity of this Smad3 mutant. Our data support an essential role of the previously uncharacterized middle region of Smad3 for nuclear functions, such as transcriptional activation and interaction with coactivators.
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Ikeda, Tohru; Kasai, Michiyuki; Tatsukawa, Eri; Kamitakahara, Masanobu; Shibata, Yasuaki; Yokoi, Taishi; Nemoto, Takayuki K; Ioku, Koji
2014-01-01
The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism. PMID:24286277
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Lv, Yanni; Qian, Yisong; Ou-Yang, Aijun; Fu, Longsheng
2016-11-01
Microglia activation initiates a neurological deficit cascade that contributes to substantial neuronal damage and impairment following ischemia stroke. Toll-like receptor 4 (TLR4) has been demonstrated to play a critical role in this cascade. In the current study, we tested the hypothesis that hydroxysafflor yellow A (HSYA), an active ingredient extracted from Flos Carthami tinctorii, alleviated inflammatory damage, and mediated neurotrophic effects in neurons by inducing the TLR4 pathway in microglia. A non-contact Transwell co-culture system comprised microglia and neurons was treated with HSYA followed by a 1 mg/mL lipopolysaccharide (LPS) stimulation. The microglia were activated prior to neuronal apoptosis, which were induced by increasing TLR4 expression in the activated microglia. However, HSYA suppressed TLR4 expression in the activated microglia, resulting in less neuronal damage at the early stage of LPS stimulation. Western blot analysis and immunofluorescence indicated that dose-dependently HSYA down-regulated TLR4-induced downstream effectors myeloid differentiation factor 88 (MyD88), nuclear factor kappa b (NF-κB), and the mitogen-activated protein kinases (MAPK)-regulated proteins c-Jun NH2-terminal protein kinase (JNK), protein kinase (ERK) 1/2 (ERK1/2), p38 MAPK (p38), as well as the LPS-induced inflammatory cytokine release. However, HSYA up-regulated brain-derived neurotrophic factor (BDNF) expression. Our data suggest that HSYA could exert neurotrophic and anti-inflammatory functions in response to LPS stimulation by inhibiting TLR4 pathway-mediated signaling.
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Rothman, S. M.; Mattson, M. P.
2013-01-01
During development of the nervous system, the formation of connections (synapses) between neurons is dependent upon electrical activity in those neurons, and neurotrophic factors produced by target cells play a pivotal role in such activity-dependent sculpting of the neural networks. A similar interplay between neurotransmitter and neurotrophic factor signaling pathways mediates adaptive responses of neural networks to environmental demands in adult mammals, with the excitatory neurotransmitter glutamate and brain-derived neurotrophic factor (BDNF) being particularly prominent regulators of synaptic plasticity throughout the central nervous system. Optimal brain health throughout the lifespan is promoted by intermittent challenges such as exercise, cognitive stimulation and dietary energy restriction, that subject neurons to activity-related metabolic stress. At the molecular level, such challenges to neurons result in the production of proteins involved in neurogenesis, learning and memory and neuronal survival; examples include proteins that regulate mitochondrial biogenesis, protein quality control, and resistance of cells to oxidative, metabolic and proteotoxic stress. BDNF signaling mediates up-regulation of several such proteins including the protein chaperone GRP-78, antioxidant enzymes, the cell survival protein Bcl-2, and the DNA repair enzyme APE1. Insufficient exposure to such challenges, genetic factors may conspire to impair BDNF production and/or signaling resulting in the vulnerability of the brain to injury and neurodegenerative disorders including Alzheimer’s, Parkinson’s and Huntington’s diseases. Further, BDNF signaling is negatively regulated by glucocorticoids. Glucocorticoids impair synaptic plasticity in the brain by negatively regulating spine density, neurogenesis and long-term potentiation, effects that are potentially linked to glucocorticoid regulation of BDNF. Findings suggest that BDNF signaling in specific brain regions mediates some of the beneficial effects of exercise and energy restriction on peripheral energy metabolism and the cardiovascular system. Collectively, the findings described in this article suggest the possibility of developing prescriptions for optimal brain health based on activity-dependent BDNF signaling. PMID:23079624
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Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas
2015-06-01
Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.
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Suryawanshi, Gajendra W.; Hoffmann, Alexander
2015-01-01
Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif’s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that target HIV-1 accessory proteins and boost antiviral activity of host factors. PMID:26385832
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Masuhara, Masaaki; Sato, Takuya; Hada, Naoto; Hakeda, Yoshiyuki
2009-01-01
Disruption of the cooperative function balance between osteoblasts and osteoclasts causes various bone disorders, some of which are attributed to abnormal osteoclast recruitment. Osteoclast differentiation is dependent on the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) as well as the macrophage colony-stimulating factor. The osteoclast formation induced by cytokines requires activation of NF-kappaB, AP-1 and nuclear factor of activated T cells c1. However, osteoclasts are not the only cell types that express these transcription factors, suggesting that some unknown molecules specific for osteoclasts may associate with the transcription factors. Here, we explored the possibility of molecules binding directly to NF-kappaB and cloned protective protein/cathepsin A (PPCA) by yeast two-hybrid screening using a cDNA library of osteoclast precursors. Forced expression of PPCA with p50/p65 in HEK293 cells decreased both the level of p50/p65 proteins and the transcriptional activity. Abundant PPCA was detected in the lysosomes of the transfected HEK293 cells, but a small amount of this enzyme was also present in the cytosolic fraction. In addition, over-expression of PPCA caused the disappearance of p50/p65 in both the lysosomal and cytosolic fractions. PPCA was expressed throughout osteoclastogenesis, and the expression was slightly up-regulated by RANKL signaling. Knockdown of PPCA in osteoclast precursors with PPCA siRNA stimulated binding of nuclear proteins to oligonucleotides containing an NF-kappaB binding motif and increased osteoclastogenesis. Our present results indicate a novel role for PPCA in osteoclastogenesis via down-regulation of NF-kappaB activity and suggest a new function for PPCA as an NF-kappaB-degrading enzyme in addition to its known multifunctional properties.
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Swamy, N; Ghosh, S; Schneider, G B; Ray, R
2001-01-01
Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal conversion of DBP to DBP-maf requires the selective removal of galactose and sialic acid from the third domain of the protein. Hence, the differential effects of the two recombinant forms of DBP-maf is most likely related to glycosylation; E. coli expressed recombinant DBP is non-glycosylated, whereas the baculovirus expressed form is glycosylated. These data support the essential role of glycosylation for the osteoclast activating property of DBP-maf. Copyright 2001 Wiley-Liss, Inc.
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Integration of growth factor signals at the c-fos serum response element.
Price, M A; Hill, C; Treisman, R
1996-04-29
A transcription factor ternary complex composed of serum response factor (SRF) and a second factor, ternary complex factor (TCF), mediates the response of the c-fos Serum Response Element to growth factors and mitogens. In NIH3T3 fibroblasts, TCF binding is required for transcriptional activation by the SRE in response to activation of the Ras-Raf-ERK pathway. We compared the properties of three members of the TCF family, Elk-1, SAP-1 and SAP-2 (ERP/NET). Although all the proteins contain sequences required for ternary complex formation with SRF, only Elk-1 and SAP-1 appear to interact with the c-fos SRE efficiently in vivo. Each TCF contains a C-terminal activation domain capable of transcriptional activation in response to activation of the Ras-Raf-ERK pathway, and this is dependent on the integrity of S/T-P motifs conserved between all the TCF family members. In contrast, activation of the SRE by whole serum and the mitogenic phospholipid LPA requires SRF binding alone. Constitutively activated members of the Rho subfamily of Ras-like GTPases are also capable of inducing activation of the SRE in the absence of TCF; unlike activated Ras itself, these proteins do not activate the TCFs in NIH3T3 cells. At the SRE, SRF- and TCF-linked signalling pathways act synergistically to potentiate transcription.
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Yacoub, Daniel; Hachem, Ahmed; Théorêt, Jean-François; Gillis, Marc-Antoine; Mourad, Walid; Merhi, Yahye
2010-12-01
CD40 ligand is a thromboinflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40 ligand (sCD40L), which has been shown to influence platelet activation, although its exact functional impact on platelets and the underlying mechanisms remain undefined. We aimed to determine the impact and the signaling mechanisms of sCD40L on platelets. sCD40L strongly enhances platelet activation and aggregation. Human platelets treated with a mutated form of sCD40L that does not bind CD40, and CD40(-/-) mouse platelets failed to elicit such responses. Furthermore, sCD40L stimulation induces the association of the tumor necrosis factor receptor-associated factor-2 with platelet CD40. Notably, sCD40L primes platelets through activation of the small GTPase Rac1 and its downstream target p38 mitogen-activated protein kinase, which leads to platelet shape change and actin polymerization. Moreover, sCD40L exacerbates thrombus formation and leukocyte infiltration in wild-type mice but not in CD40(-/-) mice. sCD40L enhances agonist-induced platelet activation and aggregation through a CD40-dependent tumor necrosis factor receptor-associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway. Thus, sCD40L is an important platelet primer predisposing platelets to enhanced thrombus formation in response to vascular injury. This may explain the link between circulating levels of sCD40L and cardiovascular diseases.
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The role of SIRT1 in diabetic retinopathy.
Karbasforooshan, Hedyieh; Karimi, Gholamreza
2018-01-01
The prevalence of diabetes mellitus (DM), has been increasing worldwide. Diabetic retinopathy (DR) is the most common microvascular complication in diabetes. It is a multifactorial disease that occurs primarily through the long-term detrimental effects of hyperglycemia. The pathogenesis of DR is complex, including inflammation, oxidative stress and advanced glycation end products (AGES). SIRT1 is a nicotinamide adenosine dinucleotide (NAD+)-dependent deacetylase that removes acetyl groups from proteins which can be implicated in DR. Inhibition of miRNAs such as miR-23b- 3P and miR-34a and activation of adenosine monophosphate-activated protein kinas (AMPK) and Peroxisome proliferative-activated receptor α (PPARα), modulate inflammation by enhancing the level of SIRT1. SIRT1 activation leads to the down regulation of nuclear factor kappa-light-chain-enhancer of activated of B cells (NF-κB), and the downstream pathway including increased level of Interleukin-17 (IL-17) and other pro-inflammatory cytokines. Oxidative stress- induced apoptosis is due to activation of some transcriptional factors such as p53 and Protein arginine methyl transferase 1 (PRMIT1) which are inhibited by SIRT1. In addition to these, the increased level of some transcriptional factors such as, vascular endothelial growth factor (VEGF), hypoxia - induced factors (HIFs), transforming growth factor β1 (TGF-β1), endothelin-1 (ET-1), fork head box O 1 (FOXO1) and Notch signaling may be inhibited by activation of SIRT1 leads to attenuation of vascular dysfunction. In conclusion, SIRT1 regulates apoptosis, inflammation and oxidative stress resulting in improving DR. This review focuses on the role of SIRT1 in DR. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
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Le Goff, Arnaud; Ji, Zongling; Leclercq, Bérénice; Bourette, Roland P.; Mougel, Alexandra; Guerardel, Cateline; de Launoit, Yvan; Vicogne, Jérôme; Goormachtigh, Gautier; Fafeur, Véronique
2012-01-01
The GRB2-associated binder 1 (GAB1) docking/scaffold protein is a key mediator of the MET-tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). Activated MET promotes recruitment and tyrosine phosphorylation of GAB1, which in turn recruits multiple proteins and mediates MET signaling leading to cell survival, motility, and morphogenesis. We previously reported that, without its ligand, MET is a functional caspase target during apoptosis, allowing the generation of a p40-MET fragment that amplifies apoptosis. In this study we established that GAB1 is also a functional caspase target by evidencing a caspase-cleaved p35-GAB1 fragment that contains the MET binding domain. GAB1 is cleaved by caspases before MET, and the resulting p35-GAB1 fragment is phosphorylated by MET upon HGF/SF binding and can interact with a subset of GAB1 partners, PI3K, and GRB2 but not with SHP2. This p35-GAB1 fragment favors cell survival by maintaining HGF/SF-induced MET activation of AKT and by hindering p40-MET pro-apoptotic function. These data demonstrate an anti-apoptotic role of caspase-cleaved GAB1 in HGF/SF-MET signaling. PMID:22915589
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Addi, Cyril; Murschel, Frédéric; Liberelle, Benoît; Riahi, Nesrine; De Crescenzo, Gregory
2017-03-01
In the field of tissue engineering, the tethering of growth factors to tissue scaffolds in an oriented manner can enhance their activity and increase their half-life. We chose to investigate the capture of the basic Fibroblast Growth Factor (bFGF) and the Epidermal Growth Factor (EGF) on a gelatin layer, as a model for the functionalization of collagen-based biomaterials. Our strategy relies on the use of two high affinity interactions, that is, the one between two distinct coil peptides as well as the one occurring between a collagen-binding domain (CBD) and gelatin. We expressed a chimeric protein to be used as an adaptor that comprises one of the coil peptides and a CBD derived from the human fibronectin. We proved that it has the ability to bind simultaneously to a gelatin substrate and to form a heterodimeric coiled-coil domain with recombinant growth factors being tagged with the complementary coil peptide. The tethering of the growth factors was characterized by ELISA and surface plasmon resonance-based biosensing. The bioactivity of the immobilized bFGF and EGF was evaluated by a human umbilical vein endothelial cell proliferation assay and a vascular smooth muscle cell survival assay. We found that the tethering of EGF preserved its mitogenic and anti-apoptotic activity. In the case of bFGF, when captured via our adaptor protein, changes in its natural mode of interaction with gelatin were observed. In an effort to functionalize collagen/gelatin-based biomaterials with growth factors, we have designed an adaptor protein corresponding to a collagen-binding domain fused to a coil peptide. In our strategy, this adaptor protein captures growth factors being tagged with the partner coil peptide in a specific, stable and oriented manner. We have found that the tethering of the Epidermal Growth Factor preserved its mitogenic and anti-apoptotic activity. In the case of the basic Fibroblast Growth Factor, the captured growth factor remained bioactive although its tethering via this adaptor protein modified its natural mode of interaction with gelatin. Altogether this strategy is easily adaptable to the simultaneous tethering of various growth factors. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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Xie, Wensheng; Pao, Christina; Graham, Taylor; Dul, Ed; Lu, Quinn; Sweitzer, Thomas D; Ames, Robert S; Li, Hu
2012-12-01
Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.
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Wilkins, Heather M; Harris, Janna L; Carl, Steven M; E, Lezi; Lu, Jianghua; Eva Selfridge, J; Roy, Nairita; Hutfles, Lewis; Koppel, Scott; Morris, Jill; Burns, Jeffrey M; Michaelis, Mary L; Michaelis, Elias K; Brooks, William M; Swerdlow, Russell H
2014-12-15
Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Lang, Charles H; Frost, Robert A
2002-05-01
The erosion of lean body mass resulting from protracted critical illness remains a significant risk factor for increased morbidity and mortality in this patient population. Previous studies have documented the well known impairment in nitrogen balance results from both an increase in muscle protein degradation as well as a decreased rate of both myofibrillar and sacroplasmic protein synthesis. This protein imbalance may be caused by an increased presence or activity of various catabolic agents, such as tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6 or glucocorticoids, or may be mediated via a decreased concentration or responsiveness to various anabolic hormones, such as growth hormone or insulin-like growth factor-I. This review focuses on recent developments pertaining to the importance of alterations in the growth hormone-insulin-like growth factor-I axis as a mechanism for the observed defects in muscle protein balance.
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Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.
Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D
2003-08-15
DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.
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Epidermal growth factor-stimulated protein phosphorylation in rat hepatocytes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Connelly, P.A.; Sisk, R.B.; Johnson, R.M.
1987-05-01
Epidermal growth factor (EGF) causes a 6-fold increase in the phosphorylation state of a cytosolic protein (pp36, M/sub r/ = 36,000, pI = 5.5) in hepatocytes isolated from fasted, male, Wistar rats. Stimulation of /sup 32/P incorporation is observed as early as 1 min following treatment of hepatocytes with EGF and is still present at 30 min after exposure to the growth factor. The phosphate incorporated into pp36 in response to EGF is located predominantly in serine but not tyrosine residues. Phosphorylation of pp36 does not occur in response to insulin or to agents which specifically activate the cAMP-dependent proteinmore » kinase (S/sub p/ -cAMPS), protein kinase C (PMA) or Ca/sup 2 +//calmodulin-dependent protein kinases (A23187) in these cells. Prior treatment of hepatocytes with the cAMP analog, S/sub p/-cAMPS, or ADP-ribosylation of N/sub i/, the inhibitory GTP-binding protein of the adenylate cyclase complex, does not prevent EGF-stimulated phosphorylation of pp36. However, as seen in other cell types, pretreatment of hepatocytes with PMA abolishes all EGF-mediated responses including phosphorylation of pp36. These results suggest that EGP specifically activates an uncharacterized, serine protein kinase in hepatocytes that is distal to the intrinsic EGF receptor tyrosine protein kinase. The rapid activation of this kinase suggests that it may play an important role in the early response of the cell to EGF.« less
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Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...
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USDA-ARS?s Scientific Manuscript database
All-trans-retinoic acid stimulates dendritic growth in hippocampal neurons within minutes by activating mitogen-activated protein kinase and mTOR and increasing dendritic translation of calcium calmodulin-dependent protein kinase II alpha and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionat...
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A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...
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Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K
2017-01-01
Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease. Copyright © 2016 American Society for Microbiology.
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Malu, Krishnakumar; Garhwal, Rahul; Pelletier, Margery G. H.; Gotur, Deepali; Halene, Stephanie; Zwerger, Monika; Yang, Zhong-Fa; Rosmarin, Alan G.; Gaines, Peter
2016-01-01
Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted ETS factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils. PMID:27342846
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Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans Peter; Turecek, Peter L
2017-01-05
BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays. Copyright © 2016 Elsevier B.V. All rights reserved.
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Inhibition of Retinoblastoma Protein Inactivation
2017-11-01
SUBJECT TERMS cell cycle, Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY...screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor, cancer, cell-cycle inhibition, activation...modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography . 3.0 ACCOMPLISHMENTS Summary: We
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Moon, Eun-Yi; Lee, Yu-Sun; Choi, Wahn Soo; Lee, Mi-Hee
2011-10-15
B-cell activating factor (BAFF) plays a role in the generation and the maintenance of mature B cells. Lipopolysaccharide (LPS) increased BAFF expression through the activation of toll-like receptor 4 (TLR4)-dependent signal transduction. Here, we investigated the mechanism of action on mouse BAFF (mBAFF) expression by cAMP production in Raw264.7 mouse macrophages. mBAFF expression was increased by the treatment with a cAMP analogue, dibutyryl-cAMP which is the activator of protein kinase A (PKA), cAMP effector protein. PKA activation was measured by the phosphorylation of cAMP-response element binding protein (CREB) on serine 133 (S133). cAMP production and CREB (S133) phosphorylation were augmented by LPS-stimulation. While mBAFF promoter activity was enhanced by the co-transfection with pS6-RSV-CREB, it was reduced by siRNA-CREB. PKA inhibitor, H-89, reduced CREB (S133) phosphorylation and mBAFF expression in control and LPS-stimulated macrophages. Another principal cAMP effector protein is cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. Epac was activated by the treatment with 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), Epac activator, as judged by the measurement of Rap1 activation. Basal level of mBAFF expression was increased by CPT treatment. LPS-stimulated mBAFF expression was also slightly enhanced by co-treatment with CPT. In addition, dibutyryl-cAMP and CPT enhanced mBAFF expression in bone marrow-derived macrophages (BMDM). With these data, it suggests that the activation of PKA and cAMP/Epac1/Rap1 pathways could be required for basal mBAFF expression, as well as being up-regulated in the TLR4-induced mBAFF expression. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.
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Colwill, K; Pawson, T; Andrews, B; Prasad, J; Manley, J L; Bell, J C; Duncan, P I
1996-01-01
Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors. Images PMID:8617202
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Childress, Catherine; Feuerbacher, Leigh A.; Phillips, Linda; Burgum, Alex
2013-01-01
Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases ltxA expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible for the anaerobic induction of ltxA RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of ltxA RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions −69 to −35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which is a repressor in Escherichia coli, functions as a direct transcriptional activator in A. actinomycetemcomitans; an mlc deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the −68 to −40 region of the leukotoxin promoter. Furthermore, we show that CRP activates ltxA expression indirectly by increasing the levels of Mlc. Analyses of Δmlc, Δihf, and Δihf Δmlc strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a Δihf Δmlc mutant still induces ltxA during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented. PMID:23475968
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NASA Astrophysics Data System (ADS)
Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan
2005-05-01
Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.
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Bereczky, Zsuzsanna; Kovács, Kitti B; Muszbek, László
2010-12-01
Protein C (PC) and protein S (PS) are vitamin K-dependent glycoproteins that play an important role in the regulation of blood coagulation as natural anticoagulants. PC is activated by thrombin and the resulting activated PC (APC) inactivates membrane-bound activated factor VIII and factor V. The free form of PS is an important cofactor of APC. Deficiencies in these proteins lead to an increased risk of venous thromboembolism; a few reports have also associated these deficiencies with arterial diseases. The degree of risk and the prevalence of PC and PS deficiency among patients with thrombosis and in those in the general population have been examined by several population studies with conflicting results, primarily due to methodological variability. The molecular genetic background of PC and PS deficiencies is heterogeneous. Most of the mutations cause type I deficiency (quantitative disorder). Type II deficiency (dysfunctional molecule) is diagnosed in approximately 5%-15% of cases. The diagnosis of PC and PS deficiencies is challenging; functional tests are influenced by several pre-analytical and analytical factors, and the diagnosis using molecular genetics also has special difficulties. Large gene segment deletions often remain undetected by DNA sequencing methods. The presence of the PS pseudogene makes genetic diagnosis even more complicated.
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Verdeguer, Francisco; Soustek, Meghan S.; Hatting, Maximilian; Blättler, Sharon M.; McDonald, Devin; Barrow, Joeva J.
2015-01-01
Mitochondrial oxidative and thermogenic functions in brown and beige adipose tissues modulate rates of energy expenditure. It is unclear, however, how beige or white adipose tissue contributes to brown fat thermogenic function or compensates for partial deficiencies in this tissue and protects against obesity. Here, we show that the transcription factor Yin Yang 1 (YY1) in brown adipose tissue activates the canonical thermogenic and uncoupling gene expression program. In contrast, YY1 represses a series of secreted proteins, including fibroblast growth factor 21 (FGF21), bone morphogenetic protein 8b (BMP8b), growth differentiation factor 15 (GDF15), angiopoietin-like 6 (Angptl6), neuromedin B, and nesfatin, linked to energy expenditure. Despite substantial decreases in mitochondrial thermogenic proteins in brown fat, mice lacking YY1 in this tissue are strongly protected against diet-induced obesity and exhibit increased energy expenditure and oxygen consumption in beige and white fat depots. The increased expression of secreted proteins correlates with elevation of energy expenditure and promotion of beige and white fat activation. These results indicate that YY1 in brown adipose tissue controls antagonistic gene expression programs associated with energy balance and maintenance of body weight. PMID:26503783
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Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel
2016-12-30
GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gα i3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Petit, Valérie; Nussbaumer, Ute; Dossenbach, Caroline; Affolter, Markus
2004-01-01
Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. We have performed a functional characterization of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and biochemical approaches, we demonstrate that Dof is a specific substrate for the two Drosophila FGFRs. After defining a minimal Dof rescue protein, we identify two regions important for Dof function in mesodermal and tracheal cell migration. The N-terminal 484 amino acids are strictly required for the interaction of Dof with the FGFRs. Upon receptor activation, tyrosine residue 515 becomes phosphorylated and recruits the phosphatase Corkscrew (Csw). Csw recruitment represents an essential step in FGF-induced cell migration and in the activation of the Ras/MAPK pathway. However, our results also indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Additional proteins binding either to the FGFRs, to Dof, or to Csw appear to be crucial for a chemotactic response. PMID:15082772
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Petit, Valérie; Nussbaumer, Ute; Dossenbach, Caroline; Affolter, Markus
2004-05-01
Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. We have performed a functional characterization of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and biochemical approaches, we demonstrate that Dof is a specific substrate for the two Drosophila FGFRs. After defining a minimal Dof rescue protein, we identify two regions important for Dof function in mesodermal and tracheal cell migration. The N-terminal 484 amino acids are strictly required for the interaction of Dof with the FGFRs. Upon receptor activation, tyrosine residue 515 becomes phosphorylated and recruits the phosphatase Corkscrew (Csw). Csw recruitment represents an essential step in FGF-induced cell migration and in the activation of the Ras/MAPK pathway. However, our results also indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Additional proteins binding either to the FGFRs, to Dof, or to Csw appear to be crucial for a chemotactic response.
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Wang, Huiling; Liu, Dajun; Cao, Peirang; Lecker, Stewart; Hu, Zhaoyong
2010-08-01
Defects in insulin/IGF-1 signaling stimulate muscle protein loss by suppressing protein synthesis and increasing protein degradation. Since an herbal compound, berberine, lowers blood levels of glucose and lipids, we proposed that it would improve insulin/IGF-1 signaling, blocking muscle protein losses. We evaluated whether berberine ameliorates muscle atrophy in db/db mice, a model of type 2 diabetes, by measuring protein synthesis and degradation in muscles of normal and db/db mice treated with or without berberine. We also examined mechanisms for berberine-induced changes in muscle protein metabolism. Berberine administration decreased protein synthesis and increased degradation in muscles of normal and db/db mice. The protein catabolic mechanism depended on berberine-stimulated expression of the E3 ubiquitin ligase, atrogin-1. Atrogin-1 not only increased proteolysis but also reduced protein synthesis by mechanisms that were independent of decreased phosphorylation of Akt or forkhead transcription factors. Impaired protein synthesis was dependent on a reduction in eIF3-f, an essential regulator of protein synthesis. Berberine impaired energy metabolism, activating AMP-activated protein kinase and providing an alternative mechanism for the stimulation of atrogin-1 expression. When we increased mitochondrial biogenesis by expressing peroxisome proliferator-activated receptor gamma coactivator-1alpha, berberine-induced changes in muscle protein metabolism were prevented. Berberine impairs muscle metabolism by two novel mechanisms. It impairs mitochonidrial function stimulating the expression of atrogin-1 without affecting phosphorylation of forkhead transcription factors. The increase in atrogin-1 not only stimulated protein degradation but also suppressed protein synthesis, causing muscle atrophy.
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Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes
NASA Technical Reports Server (NTRS)
Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)
1999-01-01
Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.
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Discovery of Curcumin, a Component of the Golden Spice, and Its Miraculous Biological Activities
Gupta, Subash C; Patchva, Sridevi; Koh, Wonil; Aggarwal, Bharat B
2012-01-01
SUMMARY 1. Curcumin is the active ingredient of the dietary spice turmeric and has been consumed for medicinal purposes for thousands of years. Modern science has shown that curcumin modulates various signaling molecules, including inflammatory molecules, transcription factors, enzymes, protein kinases, protein reductases, carrier proteins, cell survival proteins, drug resistance proteins, adhesion molecules, growth factors, receptors, cell-cycle regulatory proteins, chemokines, DNA, RNA, and metal ions. 2. Because of this polyphenol's potential to modulate multiple signaling molecules, it has been reported to possess pleiotropic activities. First shown to have anti-bacterial activity in 1949, curcumin has since been shown to have anti-inflammatory, anti-oxidant, pro-apoptotic, chemopreventive, chemotherapeutic, anti-proliferative, wound healing, anti-nociceptive, anti-parasitic, and anti-malarial properties as well. Animal studies have suggested that curcumin may be active against a wide range of human diseases, including diabetes, obesity, neurologic and psychiatric disorders, and cancer, as well as chronic illnesses affecting the eyes, lungs, liver, kidneys, and gastrointestinal and cardiovascular systems. 3. Although many clinical trials evaluating curcumin's safety and efficacy against human ailments have already been completed, others are still ongoing. Moreover, curcumin is used as a supplement in several countries, including India, Japan, the United States, Thailand, China, Korea, Turkey, South Africa, Nepal, and Pakistan. Although inexpensive, apparently well tolerated, and potentially active, curcumin has yet not been approved for treatment of any human disease. 4. In this article, we discuss the discovery and key biological activities of curcumin, with a particular emphasis on its activities at the molecular, cellular, animal, and human levels. PMID:22118895
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The role of STATs in lung carcinogenesis: an emerging target for novel therapeutics.
Karamouzis, Michalis V; Konstantinopoulos, Panagiotis A; Papavassiliou, Athanasios G
2007-05-01
The signal transducer and activator of transcription (STAT) proteins are a family of latent cytoplasmic transcription factors, which form dimers when activated by cytokine receptors, tyrosine kinase growth factor receptors as well as non-receptor tyrosine kinases. Dimeric STATs translocate to the nucleus, where they bind to specific DNA-response elements in the promoters of target genes, thereby inducing unique gene expression programs often in association with other transcription regulatory proteins. The functional consequence of different STAT proteins activation varies, as their target genes play diverse roles in normal cellular/tissue functions, including growth, apoptosis, differentiation and angiogenesis. Certain activated STATs have been implicated in human carcinogenesis, albeit only few studies have focused into their role in lung tumours. Converging evidence unravels their molecular interplays and complex multipartite regulation, rendering some of them appealing targets for lung cancer treatment with new developing strategies.
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Purification and some properties of the protein component of tissue thromboplastin from human brain.
Bjorklid, E; Storm, E
1977-01-01
The protein component of tissue thromboplastib (Factor III) from human brain was purified by extraction of a microsomal fraction with sodium deoxycholate, gel filtration of the extract on Sephadex G-100 and preparative polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The product, apoprotein III, was homogeneous by anayltical polyacrylamide-gel electrophoresis, and it induced monospecific antibodies in rabbits and goat as shown by immunodiffusion and immunoelectrophoresis. Amino acid- and carbohydrate-analysis data for apoprotein III are presented. The carbohydrate moiety of the protein consists of fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminate, amounting to a total content of 6.3g/100g. The apoprotein alone had no procoagulant activity. When Factor III was reconstituted by combining the pure apoprotein with a purified lipid fraction from the deoxycholate extract of crude Factor III, a high and optimal procoagulant activity was obtained at a phospholipid/protein ratio of 1.1g/g. Phosphatidylethanolamine alone had a weak but significant ability to restore activity, whereas phosphatidylcholine and phosphatidylserine separately had almost none. Two-component mixtures were on average more effective, and three-component mixtures far more effective, than the single phospholipids. The inclusion of a small amount of phosphatidylserine was very important for high activity. Images Fig. 2. PLATE 1 PMID:889578
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Hydrolysis of platelet-activating factor by human serum paraoxonase.
Rodrigo, L; Mackness, B; Durrington, P N; Hernandez, A; Mackness, M I
2001-01-01
Human serum paraoxonase (human PON1) has been shown to be important in the metabolism of phospholipid and cholesteryl ester hydroperoxides, thereby preventing the oxidation of low-density lipoprotein (LDL) and retarding atherogenesis. However, the exact substrate specificity of PON1 has not been established. In the present study we show that purified PON1 hydrolyses platelet-activating factor (PAF). We could find no evidence for contamination of our preparation with authentic platelet-activating-factor acetylhydrolase (PAFAH) by immunoblotting with a PAFAH monoclonal antibody or by sequencing the purified protein. In addition the specific PAFAH inhibitor SB-222657 did not affect the ability of PON1 to hydrolyse PAF (30.1+/-2.8 micromol/min per mg of protein with no inhibitor; 31.4+/-2.2 micromol/min per mg of protein with 100 nM inhibitor) or phenyl acetate (242.6+/-30.8 versus 240.8+/-31.5 micromol/min per mg of protein with and without inhibitor respectively). SB-222657 was also unable to inhibit PAF hydrolysis by isolated human high-density lipoprotein (HDL), but completely abolished the activity of human LDL. Ostrich (Struthio camelus) HDL, which does not contain PON1, was unable to hydrolyse PAF. These data provide evidence that PON1 may limit the action of this bioactive pro-inflammatory phospholipid. PMID:11171072
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Synergistic Regulation of Competence Development in Bacillus subtilis by Two Rap-Phr Systems† ‡
Bongiorni, Cristina; Ishikawa, Shu; Stephenson, Sophie; Ogasawara, Naotake; Perego, Marta
2005-01-01
The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Mol. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and RapF-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity. PMID:15968044
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Matsuo, Noritaka; Yu-Hua, Wang; Sumiyoshi, Hideaki; Sakata-Takatani, Keiko; Nagato, Hitoshi; Sakai, Kumiko; Sakurai, Mami; Yoshioka, Hidekatsu
2003-08-29
We have characterized the proximal promoter region of the human COL11A1 gene. Transient transfection assays indicate that the segment from -199 to +1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated that the ATTGG sequence, within the -147 to -121 fragment, is critical to bind nuclear proteins in the proximal COL11A1 promoter. We demonstrated that the CCAAT binding factor (CBF/NF-Y) bound to this region using an interference assay with consensus oligonucleotides and a supershift assay with specific antibodies in an EMSA. In a chromatin immunoprecipitation assay and EMSA using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We also showed that four tandem copies of the CBF/NF-Y-binding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a CBF/NF-Y-binding fragment suppressed the COL11A1 promoter activity. Furthermore, overexpression of a dominant-negative CBF-B/NF-YA subunit significantly inhibited promoter activity in both transient and stable cells. These results indicate that the CBF/NF-Y proteins regulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter region.
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Itoh, Motoyuki; Yoshida, Yuichi; Nishida, Keigo; Narimatsu, Masahiro; Hibi, Masahiko; Hirano, Toshio
2000-01-01
Gab1 is a member of the Gab/DOS (Daughter of Sevenless) family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for SH2 and SH3 domains. Gab1 is tyrosine phosphorylated upon stimulation of various cytokines, growth factors, and antigen receptors in cell lines and interacts with signaling molecules, such as SHP-2 and phosphatidylinositol 3-kinase, although its biological roles have not yet been established. To reveal the functions of Gab1 in vivo, we generated mice lacking Gab1 by gene targeting. Gab1-deficient embryos died in utero and displayed developmental defects in the heart, placenta, and skin, which were similar to phenotypes observed in mice lacking signals of the hepatocyte growth factor/scatter factor, platelet-derived growth factor, and epidermal growth factor pathways. Consistent with these observations, extracellular signal-regulated kinase mitogen-activated protein (ERK MAP) kinases were activated at much lower levels in cells from Gab1-deficient embryos in response to these growth factors or to stimulation of the cytokine receptor gp130. These results indicate that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAP kinase activation. PMID:10779359
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Li, Hongbo; Gao, Xuefei; Zhou, Yi; Li, Na; Ge, Caozuo; Hui, Xiaoyan; Wang, Yu; Xu, Aimin; Jin, Shouguang; Wu, Donghai
2011-09-01
C1q and tumor necrosis factor related proteins (CTRPs) are a family of adiponectin paralogues. Among them, CTRP2 is the only CTRP protein that has been shown to possess similar biological activities as adiponectin. To further explore the physiological roles of human CTRP2 and its mechanisms of action, hCTRP2 gene was expressed in Escherichia coli and Pichia pastoris, respectively. In the P. pastoris expression system, recombinant hCTRP2 could be secreted into the culture medium under induction condition, however, the resultant recombinant protein was highly unstable, resulting two main degradation products with molecular masses of approximately 20 and 26 kDa, respectively. In the E. coli expression system, a large amount of soluble thioredoxin (Trx)-hCTRP2 fusion protein could be produced, which accounts about 42% of the total soluble bacterial proteins. The recombinant Trx-hCTRP2 fusion protein was purified to an approximately 95% purity using Ni-NTA affinity chromatography and Superdex G-75 column with a yield of about 15 mg/l protein from 1l bacterial culture. The purified recombinant Trx-hCTRP2 was shown to be active under in vitro assay conditions. Copyright © 2011 Elsevier Inc. All rights reserved.
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Sosnowska, Danuta; Richardson, Chris; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan; Ridgway, Iain
2014-12-01
Study of negligibly senescent animals may provide clues that lead to better understanding of the cardiac aging process. To elucidate mechanisms of successful cardiac aging, we investigated age-related changes in proteasome activity, oxidative protein damage and expression of heat shock proteins, inflammatory factors, and mitochondrial complexes in the heart of the ocean quahog Arctica islandica, the longest-lived noncolonial animal (maximum life span potential: 508 years). We found that in the heart of A. islandica the level of oxidatively damaged proteins did not change significantly up to 120 years of age. No significant aging-induced changes were observed in caspase-like and trypsin-like proteasome activity. Chymotrypsin-like proteasome activity showed a significant early-life decline, then it remained stable for up to 182 years. No significant relationship was observed between the extent of protein ubiquitination and age. In the heart of A. islandica, an early-life decline in expression of HSP90 and five mitochondrial electron transport chain complexes was observed. We found significant age-related increases in the expression of three cytokine-like mediators (interleukin-6, interleukin-1β, and tumor necrosis factor-α) in the heart of A. islandica. Collectively, in extremely long-lived molluscs, maintenance of protein homeostasis likely contributes to the preservation of cardiac function. Our data also support the concept that low-grade chronic inflammation in the cardiovascular system is a universal feature of the aging process, which is also manifest in invertebrates. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Abbott, Jason R; Hodges, Timothy R; Daniels, R Nathan; Patel, Pratiq A; Kennedy, Jack Phillip; Howes, Jennifer E; Akan, Denis T; Burns, Michael C; Sai, Jiqing; Sobolik, Tammy; Beesetty, Yugandhar; Lee, Taekyu; Rossanese, Olivia W; Phan, Jason; Waterson, Alex G; Fesik, Stephen W
2018-06-01
Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure-activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at sub-micromolar concentrations in vitro, increase levels of active RAS-GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway, resulting in a decrease in pERK1/2 T202/Y204 protein levels at higher compound concentrations.
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Heparin Microparticle Effects on Presentation and Bioactivity of Bone Morphogenetic Protein-2
Hettiaratchi, Marian H.; Miller, Tobias; Temenoff, Johnna S.; Guldberg, Robert E.; McDevitt, Todd C.
2014-01-01
Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration. PMID:24881028
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NASA Technical Reports Server (NTRS)
Thomas, M. J.; Umayahara, Y.; Shu, H.; Centrella, M.; Rotwein, P.; McCarthy, T. L.
1996-01-01
Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.
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Chen, Meng-Chuan; Lee, Chi-Feng; Huang, Wen-Hsin; Chou, Tz-Chong
2013-05-01
The hypoxic environment in tumors is an important factor causing tumor angiogenesis by activating the key transcription factor, hypoxia-inducible factors-1α (HIF-1α). Magnolol isolated from Magnolia officinalis has been reported to exhibit an anticancer activity via elevation of apoptosis. However, whether magnolol inhibits tumor angiogenesis remains unknown. In the present study, we demonstrated that magnolol significantly inhibited angiogenesis in vitro and in vivo evidenced by the attenuation of hypoxia and vascular endothelial growth factor (VEGF)-induced tube formation of human umbilical vascular endothelial cells, vasculature generation in chicken chorioallantoic membrane and Matrigel plug. In hypoxic human bladder cancer cells (T24), treatment with magnolol inhibited hypoxia-stimulated H2O2 formation, HIF-1α induction including mRNA, protein expression, and transcriptional activity as well as VEGF secretion. Additionally, the enhanced degradation of HIF-1α protein via enhancing prolyl hydroxylase activity and the decreased newly-synthesized HIF-1α protein in hypoxic T24 cells may involve the reduction of HIF-1α protein accumulation by magnolol. Interestingly, magnolol also acts as a VEGFR2 antagonist, and subsequently attenuates the down-stream AKT/mTOR/p70S6K/4E-BP-1 kinase activation both in hypoxic T24 cells and tumor tissues. As expected, administration of magnolol greatly attenuated tumor growth, angiogenesis and the protein expression of HIF-1α, VEGF, CD31, a marker of endothelial cells, and carbonic anhydrase IX, an endogenous marker for hypoxia, in the T24 xenograft mouse model. Collectively, these findings strongly indicate that the anti-agngiogenic activity of magnolol is, at least in part, mediated by suppressing HIF-1α/VEGF-dependent pathways, and suggest that magnolol may be a potential drug for human bladder cancer therapy. Copyright © 2013 Elsevier Inc. All rights reserved.
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Touati, Sabeur; Montezano, Augusto C I; Meziri, Fayçal; Riva, Catherine; Touyz, Rhian M; Laurant, Pascal
2015-02-01
Exercise training reverses atherosclerotic risk factors associated with metabolic syndrome and obesity. The aim of the present study was to determine the molecular anti-inflammatory, anti-oxidative and anti-atherogenic effects in aorta from rats with high-fat diet-induced obesity. Male Sprague-Dawley rats were placed on a high-fat (HFD) or control (CD) diet for 12 weeks. The HFD rats were then divided into four groups: (i) sedentary HFD-fed rats (HFD-S); (ii) exercise trained (motor treadmill 5 days/week, 60 min/day, 12 weeks) HFD-fed rats (HFD-Ex); (iii) modified diet (HFD to CD) sedentary rats (HF/CD-S); and (iv) an exercise-trained modified diet group (HF/CD-Ex). Tissue levels of NADPH oxidase (activity and expression), NADPH oxidase (Nox) 1, Nox2, Nox4, p47(phox) , superoxide dismutase (SOD)-1, angiotensin AT1 and AT2 receptors, phosphorylated mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the aorta. Plasma cytokines (tumour necrosis factor (TNF)-α and interleukin (IL)-6) levels were also measured. Obesity was accompanied by increases in NADPH oxidase activity, p47(phox) translocation, Nox4 and VCAM-1 protein expression, MAPK (ERK1/2, SAPK/JNK) phosphorylation and plasma TNF-α and IL-6 levels. Exercise training and switching from the HFD to CD reversed almost all these molecular changes. In addition, training increased aortic SOD-1 protein expression and decreased ERK1/2 phosphorylation. These findings suggest that protective effects of exercise training on atherosclerotic risk factors induced by obesity are associated with downregulation of NADPH oxidase, ERK1/2 and SAPK/JNK activity and increased SOD-1 expression. © 2014 Wiley Publishing Asia Pty Ltd.
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The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.
Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D
1993-05-06
Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.
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León-López, Liliana; Márquez-Mota, Claudia C; Velázquez-Villegas, Laura A; Gálvez-Mariscal, Amanda; Arrieta-Báez, Daniel; Dávila-Ortiz, Gloria; Tovar, Armando R; Torres, Nimbe
2015-09-01
Jatropha curcas is an oil seed plant that belongs to the Euphorbiaceae family. Nontoxic genotypes have been reported in Mexico. The purpose of the present work was to evaluate the effect of a Mexican variety of J. curcas protein concentrate (JCP) on weight gain, biochemical parameters, and the expression of genes and proteins involved in insulin signaling, lipogenesis, cholesterol and protein synthesis in rats. The results demonstrated that short-term consumption of JCP increased serum glucose, insulin, triglycerides and cholesterol levels as well as the expression of transcription factors involved in lipogenesis and cholesterol synthesis (SREBP-1 and LXRα). Moreover, there was an increase in insulin signaling mediated by Akt phosphorylation and mTOR. JCP also increased PKCα protein abundance and the activation of downstream signaling pathway targets such as the AP1 and NF-κB transcription factors typically activated by phorbol esters. These results suggested that phorbol esters are present in JCP, and that they could be involved in the activation of PKC which may be responsible for the high insulin secretion and consequently the activation of insulin-dependent pathways. Our data suggest that this Mexican Jatropha variety contains toxic compounds that produce negative metabolic effects which require caution when using in the applications of Jatropha-based products in medicine and nutrition.
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TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures
NASA Astrophysics Data System (ADS)
Feller, Georges
2010-08-01
Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.
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Factor VIII organisation on nanodiscs with different lipid composition.
Grushin, Kirill; Miller, Jaimy; Dalm, Daniela; Stoilova-McPhie, Svetla
2015-04-01
Nanodiscs (ND) are lipid bilayer membrane patches held by amphiphilic scaffolding proteins (MSP) of ~10 nm in diameter. Nanodiscs have been developed as lipid nanoplatforms for structural and functional studies of membrane and membrane associated proteins. Their size and monodispersity have rendered them unique for electron microscopy (EM) and single particle analysis studies of proteins and complexes either spanning or associated to the ND membrane. Binding of blood coagulation factors and complexes, such as the Factor VIII (FVIII) and the Factor VIIIa - Factor IXa (intrinsic tenase) complex to the negatively charged activated platelet membrane is required for normal haemostasis. In this study we present our work on optimising ND, specifically designed to bind FVIII at close to physiological conditions. The binding of FVIII to the negatively charged ND rich in phosphatidylserine (PS) was followed by electron microscopy at three different PS compositions and two different membrane scaffolding protein (MSP1D1) to lipid ratios. Our results show that the ND with highest PS content (80 %) and lowest MSP1D1 to lipid ratio (1:47) are the most suitable for structure determination of the membrane-bound FVIII by single particle EM. Our preliminary FVIII 3D reconstruction as bound to PS containing ND demonstrates the suitability of the optimised ND for structural studies by EM. Further assembly of the activated FVIII form (FVIIIa) and the whole FVIIIa-FIXa complex on ND, followed by EM and single particle reconstruction will help to identify the protein-protein and protein-membrane interfaces critical for the intrinsic tenase complex assembly and function.
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Genetic and pharmacological suppression of oncogenic mutations in RAS genes of yeast and humans
DOE Office of Scientific and Technical Information (OSTI.GOV)
Schafer, W.R.; Sterne, R.; Thorner, J.
1989-07-28
The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast-a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay.more » The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations. 50 refs., 3 figs., 3 tabs.« less
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Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L
2014-10-16
Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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Agarwal, Pratul K.
2015-11-24
A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.
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Agarwal, Pratul K.
2013-04-09
A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.
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ERRα protein is stabilized by LSD1 in a demethylation-independent manner.
Carnesecchi, Julie; Cerutti, Catherine; Vanacker, Jean-Marc; Forcet, Christelle
2017-01-01
The LSD1 histone demethylase is highly expressed in breast tumors where it constitutes a factor of poor prognosis and promotes traits of cancer aggressiveness such as cell invasiveness. Recent work has shown that the Estrogen-Related Receptor α (ERRα) induces LSD1 to demethylate the Lys 9 of histone H3. This results in the transcriptional activation of a number of common target genes, several of which being involved in cellular invasion. High expression of ERRα protein is also a factor of poor prognosis in breast tumors. Here we show that, independently of its demethylase activities, LSD1 protects ERRα from ubiquitination, resulting in overexpression of the latter protein. Our data also suggests that the elevation of LSD1 mRNA and protein in breast cancer (as compared to normal tissue) may be a key event to increase ERRα protein, independently of its corresponding mRNA.
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ERRα protein is stabilized by LSD1 in a demethylation-independent manner
Carnesecchi, Julie; Cerutti, Catherine; Vanacker, Jean-Marc
2017-01-01
The LSD1 histone demethylase is highly expressed in breast tumors where it constitutes a factor of poor prognosis and promotes traits of cancer aggressiveness such as cell invasiveness. Recent work has shown that the Estrogen-Related Receptor α (ERRα) induces LSD1 to demethylate the Lys 9 of histone H3. This results in the transcriptional activation of a number of common target genes, several of which being involved in cellular invasion. High expression of ERRα protein is also a factor of poor prognosis in breast tumors. Here we show that, independently of its demethylase activities, LSD1 protects ERRα from ubiquitination, resulting in overexpression of the latter protein. Our data also suggests that the elevation of LSD1 mRNA and protein in breast cancer (as compared to normal tissue) may be a key event to increase ERRα protein, independently of its corresponding mRNA. PMID:29190800
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Xu, Bingfang; Abdel-Fattah, Rana; Yang, Ling; Crenshaw, Sallie A.; Black, Michael B.; Hinton, Barry T.
2011-01-01
The initial segment of the epididymis is vital for male fertility; therefore, it is important to understand the mechanisms that regulate this important region. Deprival of testicular luminal fluid factors/lumicrine factors from the epididymis results in a wave of apoptosis in the initial segment. In this study, a combination of protein array and microarray analyses was used to examine the early changes in downstream signal transduction pathways following loss of lumicrine factors. We discovered the following cascade of events leading to the loss of protection and eventual apoptosis: in the first 6 h after loss of lumicrine factors, down-regulation of the ERK pathway components was observed at the mRNA expression and protein activity levels. Microarray analysis revealed that mRNA levels of several key components of the ERK pathway, Dusp6, Dusp5, and Etv5, decreased sharply, while the analysis from the protein array revealed a decline in the activities of MAP2K1/2 and MAPK1. Immunostaining of phospho-MAPK3/1 indicated that down-regulation of the ERK pathway was specific to the epithelial cells of the initial segment. Subsequently, after 12 h of loss of lumicrine factors, levels of mRNA expression of STAT and NFKB pathway components increased, mRNA levels of several genes encoding cell cycle inhibitors increased, and levels of protein expression of several proapoptotic phosphatases increased. Finally, after 18 h of loss of protection from lumicrine factors, apoptosis was observed. In conclusion, testicular lumicrine factors protect the cells of the initial segment by activating the ERK pathway, repressing STAT and NFKB pathways, and thereby preventing apoptosis. PMID:21311037
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Growth factor involvement in tension-induced skeletal muscle growth
NASA Technical Reports Server (NTRS)
Vandenburgh, Herman H.
1993-01-01
Long-term manned space travel will require a better understanding of skeletal muscle atrophy which results from microgravity. Astronaut strength and dexterity must be maintained for normal mission operations and for emergency situations. Although exercise in space slows the rate of muscle loss, it does not prevent it. A biochemical understanding of how gravity/tension/exercise help to maintain muscle size by altering protein synthesis and/or degradation rate should ultimately allow pharmacological intervention to prevent muscle atrophy in microgravity. The overall objective is to examine some of the basic biochemical processes involved in tension-induced muscle growth. With an experimental in vitro system, the role of exogenous and endogenous muscle growth factors in mechanically stimulated muscle growth are examined. Differentiated avian skeletal myofibers can be 'exercised' in tissue culture using a newly developed dynamic mechanical cell stimulator device which simulates different muscle activity patterns. Patterns of mechanical activity which significantly affect muscle growth and metabolic characteristics were found. Both exogenous and endogenous growth factors are essential for tension-induced muscle growth. Exogenous growth factors found in serum, such as insulin, insulin-like growth factors, and steroids, are important regulators of muscle protein turnover rates and mechanically-induced muscle growth. Endogenous growth factors are synthesized and released into the culture medium when muscle cells are mechanically stimulated. At least one family of mechanically induced endogenous factors, the prostaglandins, help to regulate the rates of protein turnover in muscle cells. Endogenously synthesized IGF-1 is another. The interaction of muscle mechanical activity and these growth factors in the regulation of muscle protein turnover rates with our in vitro model system is studied.
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Fragale, Alessandra; Tartaglia, Marco; Wu, Jie; Gelb, Bruce D
2004-03-01
Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation. Copyright 2004 Wiley-Liss, Inc.
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Quercetin, not caffeine, is a major neuroprotective component in coffee.
Lee, Moonhee; McGeer, Edith G; McGeer, Patrick L
2016-10-01
Epidemiologic studies indicate that coffee consumption reduces the risk of Parkinson's disease and Alzheimer's disease. To determine the factors involved, we examined the protective effects of coffee components. The test involved prevention of neurotoxicity to SH-SY5Y cells that was induced by lipopolysaccharide plus interferon-γ or interferon-γ released from activated microglia and astrocytes. We found that quercetin, flavones, chlorogenic acid, and caffeine protected SH-SY5Y cells from these toxins. They also reduced the release of tumor necrosis factor-α and interleukin-6 from the activated microglia and astrocytes and attenuated the activation of proteins from P38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFκB). After exposure to toxin containing glial-stimulated conditioned medium, we also found that quercetin reduced oxidative/nitrative damage to DNA, as well as to the lipids and proteins of SH-SY5Y cells. There was a resultant increase in [GSH]i in SH-SY5Y cells. The data indicate that quercetin is the major neuroprotective component in coffee against Parkinson's disease and Alzheimer's disease. Copyright © 2016 Elsevier Inc. All rights reserved.
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Molecular mechanisms of the impact of smoke-oxidants.
Milnerowicz, Halina; Ściskalska, Milena; Dul, Magdalena
2015-01-01
Tobacco smoke is a source of many xenobiotics and free radicals. Reactive oxygen species can affect the body both directly and indirectly, through the activation of both signalling pathways and transcription factors (NF-κB and AP-1). One of the most important signalling cascades which can affect the oxidants in smoke are mitogen-activated protein kinases (MAPK). The mechanism of MAPK pathways activation by reactive oxygen species depends on the stimulation of specific tyrosine kinases and protein tyrosine phosphatases inactivation. An activated MAP protein can initiate AP-1 signalling and interact with many other transcription factors. The components of tobacco smoke with oxidation-reduction properties can have an effect on NF-κB signalling. Binding of NF-κB and AP-1 with DNA is a complicated process, in which coactivators exhibiting internal histone acetyltransferase activity are involved. The balance between histone deacetylases and acetylases is important for the regulation of inflammatory response in the lungs. Tobacco smoke causes increased acetylase activity and decreased deacetylase activity in epithelial lung cells. The result is an increase in the activation of NF-κB and AP-1. Oxygen free radicals from tobacco smoke can change the redox status of cells, which can in turn induce the activation of transcription factors, chromatin remodelling and intensified genes transcription for inflammatory mediators. Copyright © 2015 Elsevier GmbH. All rights reserved.
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Nitric oxide - an activating factor of adenosine deaminase 2 in vitro.
Sargisova, Ye G; Andreasyan, N A; Hayrapetyan, H L; Harutyunyan, H A
2012-01-01
In this study we have investigated the effect of reactive oxygen species produced by some chemicals in aqueous solutions on activity of adenosine deaminase 2 (ADA2) purified from human blood plasma. An activating effect on ADA2 was observed in vitro with sodium nitroprusside (SNP), the source of NO (nitrosonium ions NO(-) in aqueous solutions). Not SH-groups of cysteine but other amino acid residues sensitive to NO were responsible for ADA2 activation. The SNP-derived activation was more pronounced when purified ADA2 was preincubated with heparin and different proteins as an experimental model of the protein environment in vivo. The most effective was heparin, which is known for its ability to regulate enzyme and protein functions in extracellular matrix. We conclude that ADA2 is a protein with flexible conformation that is affected by the protein environment, and it changes its activity under oxidative (nitrosative) stress.
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Thakur, Shweta; Sarkar, Bibekananda; Cholia, Ravi P; Gautam, Nandini; Dhiman, Monisha; Mantha, Anil K
2014-01-01
Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a ‘hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed. PMID:25033834
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Liang, H; Olejniczak, E T; Mao, X; Nettesheim, D G; Yu, L; Thompson, C B; Fesik, S W
1994-01-01
The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins. Images PMID:7972119
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Hale, T K; Braithwaite, A W
1999-08-20
Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.
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Glucose ingestion stimulates atherothrombotic inflammation in polycystic ovary syndrome
Kirwan, John P.; Rote, Neal S.; Minium, Judi
2013-01-01
Women with polycystic ovary syndrome (PCOS) have chronic low-grade inflammation that can increase the risk of atherothrombosis. We performed a cross-sectional study to examine the effect of glucose ingestion on markers of atherothrombotic inflammation in mononuclear cells (MNC) of 16 women with PCOS (8 lean, 8 obese) and 16 weight-matched controls. Activator protein-1 (AP-1) activation and the protein content of early growth response-1 (EGR-1), matrix matalloproteinases-2 (MMP2), and tissue factor (TF) were quantified from MNC obtained from blood drawn fasting and 2 h after glucose ingestion. Plasma MMP9 and C-reactive protein (CRP) were measured from fasting blood samples. Truncal fat was determined by DEXA. Lean women with PCOS exhibited greater AP-1 activation and MMP2 protein content after glucose ingestion and higher plasma MMP9 and CRP levels than lean controls. Obese women with PCOS exhibited greater EGR-1 and TF protein content after glucose ingestion, and plasma CRP levels were even higher compared with lean subjects regardless of PCOS status. Truncal fat correlated with MMP9 and CRP levels and glucose-stimulated increases in AP-1 activation and EGR-1 and TF protein content. Testosterone correlated with glucose-stimulated AP-1 activation, and androstenedione correlated with MMP9 and CRP levels and glucose-stimulated AP-1 activation. Thus, both PCOS and obesity contribute to an atherothrombotic state in which excess abdominal adiposity and hyperandrogenism may be specific risk factors for developing atherothrombosis. PMID:23249695
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Ko, Su Hyuk; Jeon, Jong Ik; Myung, Hyun Soo; Kim, Young-Jeon; Kim, Jung Mogg
2017-10-01
Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. Copyright © 2017 American Society for Microbiology.
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Piirsoo, Alla; Kasak, Lagle; Kauts, Mari-Liis; Loog, Mart; Tints, Kairit; Uusen, Piia; Neuman, Toomas; Piirsoo, Marko
2014-04-01
Observations that Glioma-associated transcription factors Gli1 and Gli2 (Gli1/2), executers of the Sonic Hedgehog (Shh) signaling pathway and targets of the Transforming Growth Factor β (TGF-β) signaling axis, are involved in numerous developmental and pathological processes unveil them as attractive pharmaceutical targets. Unc-51-like serine/threonine kinase Ulk3 has been suggested to play kinase activity dependent and independent roles in the control of Gli proteins in the context of the Shh signaling pathway. This study aimed at investigating whether the mechanism of generation of Gli1/2 transcriptional activators has similarities regardless of the signaling cascade evoking their activation. We also elucidate further the role of Ulk3 kinase in regulation of Gli1/2 proteins and examine SU6668 as an inhibitor of Ulk3 catalytic activity and a compound targeting Gli1/2 proteins in different cell-based experimental models. Here we demonstrate that Ulk3 is required not only for maintenance of basal levels of Gli1/2 proteins but also for TGF-β or Shh dependent activation of endogenous Gli1/2 proteins in human adipose tissue derived multipotent stromal cells (ASCs) and mouse immortalized progenitor cells, respectively. We show that cultured ASCs possess the functional Shh signaling axis and differentiate towards osteoblasts in response to Shh. Also, we demonstrate that similarly to Ulk3 RNAi, SU6668 prevents de novo expression of Gli1/2 proteins and antagonizes the Gli-dependent activation of the gene expression programs induced by either Shh or TGF-β. Our data suggest SU6668 as an efficient inhibitor of Ulk3 kinase allowing manipulation of the Gli-dependent transcriptional outcome. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.
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Kuo, Chun-Ting; Chang, Chieh; Lee, Wen-Sen
2015-01-01
To investigate the molecular mechanism underlying folic acid (FA)-induced anti-colon caner activity, we showed that FA caused G0/G1 arrest in COLO-205. FA activated the proto-oncogene tyrosine-protein kinase Src (c-SRC)-mediated signaling pathway to enhance nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) nuclear translocation and binding onto the tumor protein p53 (TP53) gene promoter, and up-regulated expressions of TP53, cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). Knock-down of TP53 abolished FA-induced increases in the levels of CDKN1A and CDKN1B protein and G0/G1 arrest in COLO-205. Knock-down of folate receptor alpha (FRα) abolished FA-induced activations in the c-SRC-mediated pathway and increases in the levels of CDKN1A, CDKN1B and TP53 protein. These data suggest that FA inhibited COLO-205 proliferation through activating the FRα/c-SRC/mitogen-activated protein kinase 3/1 (ERK1/2)/NFκB/TP53 pathway-mediated up-regulations of CDKN1A and CDKN1B protein. In vivo studies demonstrated that daily i.p. injections of FA led to profound regression of the COLO-205 tumors and prolong the lifespan. In these tumors, the levels of CDKN1A, CDKN1B and TP53 protein were increased and von willebrand factor (VWF) protein levels were decreased. These findings suggest that FA inhibits COLO-205 colon cancer growth through anti-cancer cell proliferation and anti-angiogenesis. PMID:26056802
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Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.
Rabe, Shahrzad Zamani Taghizadeh; Ghazanfari, Tooba; Siadat, Zahra; Rastin, Maryam; Rabe, Shahin Zamani Taghizadeh; Mahmoudi, Mahmoud
2015-04-01
Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities. The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages. J774A.1 macrophages were treated with 14-kDa protein (5-30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot. Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein. Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.
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Lively, T N; Ferguson, H A; Galasinski, S K; Seto, A G; Goodrich, J A
2001-07-06
c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.
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Komoike, Yuta; Matsuoka, Masato
2013-10-15
Tributyltin (TBT) is a major marine contaminant and causes endocrine disruption, hepatotoxicity, immunotoxicity, and neurotoxicity. However, the molecular mechanisms underlying the toxicity of TBT have not been fully elucidated. We examined whether exposure to TBT induces the endoplasmic reticulum (ER) stress response in zebrafish, a model organism. Zebrafish-derived BRF41 fibroblast cells were exposed to 0.5 or 1 μM TBT for 0.5-16 h and subsequently lysed and immunoblotted to detect ER stress-related proteins. Zebrafish embryos, grown until 32 h post fertilization (hpf), were exposed to 1 μM TBT for 16 h and used in whole mount in situ hybridization and immunohistochemistry to visualize the expression of ER chaperones and an ER stress-related apoptosis factor. Exposure of the BRF41 cells to TBT caused phosphorylation of the zebrafish homolog of protein kinase RNA-activated-like ER kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), and inositol-requiring enzyme 1 (IRE1), characteristic splicing of X-box binding protein 1 (XBP1) mRNA, and enhanced expression of activating transcription factor 4 (ATF4) protein. In TBT-exposed zebrafish embryos, ectopic expression of the gene encoding zebrafish homolog of the 78 kDa glucose-regulating protein (GRP78) and gene encoding CCAAT/enhancer-binding protein homologous protein (CHOP) was detected in the precursors of the neuromast, which is a sensory organ for detecting water flow and vibration. Our in vitro and in vivo studies revealed that exposure of zebrafish to TBT induces the ER stress response via activation of both the PERK-eIF2α and IRE1-XBP1 pathways of the unfolded protein response (UPR) in an organ-specific manner. Copyright © 2013 Elsevier B.V. All rights reserved.
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Wilson, Fiona A; Orellana, Renán A; Suryawan, Agus; Nguyen, Hanh V; Jeyapalan, Asumthia S; Frank, Jason; Davis, Teresa A
2008-07-01
Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7-10 days of pST (150 microg x kg(-1) x day(-1)) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 microU/ml), 2) fed control (25 microU/ml), and 3) fed pST-treated (50 microU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1.eIF4E complex association and increased active eIF4E.eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation.
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Wilson, Fiona A.; Orellana, Renán A.; Suryawan, Agus; Nguyen, Hanh V.; Jeyapalan, Asumthia S.; Frank, Jason; Davis, Teresa A.
2008-01-01
Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7–10 days of pST (150 μg·kg−1·day−1) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 μU/ml), 2) fed control (25 μU/ml), and 3) fed pST-treated (50 μU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1·eIF4E complex association and increased active eIF4E·eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation. PMID:18460595
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WRKY Proteins: Signaling and Regulation of Expression during Abiotic Stress Responses
Banerjee, Aditya
2015-01-01
WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research. PMID:25879071
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WRKY proteins: signaling and regulation of expression during abiotic stress responses.
Banerjee, Aditya; Roychoudhury, Aryadeep
2015-01-01
WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research.
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Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold
DOE Office of Scientific and Technical Information (OSTI.GOV)
Page, Michael J.; Di Cera, Enrico; St. Louis-MED)
2010-06-14
Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na{sup +}-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional proteinmore » domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.« less
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Shehzad, Adeeb; Parveen, Sajida; Qureshi, Munibah; Subhan, Fazli; Lee, Young Sup
2018-03-01
Epidemiological studies have shown that inflammation plays a critical role in the development and progression of various chronic diseases, including cancers, neurological diseases, hepatic fibrosis, diabetic retinopathy, and vascular diseases. Decursin and decursinol angelate (DA) are pyranocoumarin compounds obtained from the roots of Angelica gigas. Several studies have described the anti-inflammatory effects of decursin and DA. Decursin and DA have shown potential anti-inflammatory activity by modulating growth factors such as vascular endothelial growth factor, transcription factors such as signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells, cellular enzymes including matrix metalloproteinases cyclooxygenase, and protein kinases such as extracellular receptor kinase, phosphatidylinositol-3-kinase, and protein kinase C. These compounds have the ability to induce apoptosis by activating pro-apoptotic proteins and the caspase cascade, and reduced the expression of anti-apoptotic proteins such as B-cell lymphoma 2 and B-cell lymphoma-extra-large. Interaction with multiple molecular targets and cytotoxic effects, these two compounds are favorable candidates for treating various chronic inflammatory diseases such as cancers (prostate, breast, leukemia, cervical, and myeloma), rheumatoid arthritis, diabetic retinopathy, hepatic fibrosis, osteoclastogenesis, allergy, and Alzheimer's disease. We have summarized the preliminary studies regarding the biological effects of decursin and DA. In this review, we will also highlight the functions of coumarin compounds that can be translated to a clinical practice for the treatment and prevention of various inflammatory ailments.
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Kawamata, Yuji; Imamura, Takeshi; Babendure, Jennie L; Lu, Juu-Chin; Yoshizaki, Takeshi; Olefsky, Jerrold M
2007-09-28
Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.
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Li, Jun; Mahdi, Fakhri; Du, Lin; Datta, Sandipan; Nagle, Dale G.; Zhou, Yu-Dong
2011-01-01
Over 20000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 µM and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods. PMID:21875114
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Wolf, Sabine; Janzen, Annette; Vékony, Nicole; Martiné, Ursula; Strand, Dennis; Closs, Ellen I
2002-06-15
Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230-236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional.
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Wolf, Sabine; Janzen, Annette; Vékony, Nicole; Martiné, Ursula; Strand, Dennis; Closs, Ellen I
2002-01-01
Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230-236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional. PMID:12049641
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Kim, Maengjo; Hunter, Roger W; Garcia-Menendez, Lorena; Gong, Guohua; Yang, Yu-Ying; Kolwicz, Stephen C; Xu, Jason; Sakamoto, Kei; Wang, Wang; Tian, Rong
2014-03-14
AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory γ2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target. We sought to identify the pro-growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage. In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate-stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor-signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth. Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of γ2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.
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Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling
2014-01-01
Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046
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Lin, L; Zheng, Y; Qu, J; Bao, G
2000-06-01
Observe the effect of tumor necrosis factor-alpha (TNF-alpha) on calcium-activated potassium channel in ECV304 and the possible involvement of G-protein mediation in the action of TNF-alpha. Using the cell-attached configuration of patch clamp technique. (1) the activity of high-conductance calcium-activated potassium channel (BKca) was recorded. Its conductance is (202.54 +/- 16.62) pS; (2) the activity of BKca was potentiated by 200 U/ml TNF-alpha; (3) G-protein would intensify this TNF-alpha activation. TNF-alpha acted on vascular endothelial cell ECV304 could rapidly activate the activity of BKca. Opening of BKca resulted in membrane hyper-polarization which could increase electro-chemical gradient for the resting Ca2+ influx and open leakage calcium channel, thus resting cytoplasmic free Ca2+ concentration could be elevated. G-protein may exert an important regulation in this process.
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Sankhala, Rajeshwer S.; Damai, Rajani S.; Swamy, Musti J.
2011-01-01
The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109. PMID:21408153
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BAR domain proteins regulate Rho GTPase signaling.
Aspenström, Pontus
2014-01-01
BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.
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Yang, Yi; Li, Wang; Liu, Yang; Sun, Yuning; Li, Yan; Yao, Qing; Li, Jianning; Zhang, Qian; Gao, Yujing; Gao, Ling; Zhao, Jiajun
2014-11-01
Understanding the mechanism by which alpha-lipoic acid supplementation has a protective effect upon nonalcoholic fatty liver disease in vivo and in vitro may lead to targets for preventing hepatic steatosis. Male C57BL/6J mice were fed a normal diet, high-fat diet or high-fat diet supplemented with alpha-lipoic acid for 24 weeks. HepG2 cells were incubated with normal medium, palmitate or alpha-lipoic acid. The lipid-lowering effects were measured. The protein expression and distribution were analyzed by Western blot, immunoprecipitation and immunofluorescence, respectively. We found that alpha-lipoic acid enhanced sirtuin 1 deacetylase activity through liver kinase B1 and stimulated AMP-activated protein kinase. By activating the sirtuin 1/liver kinase B1/AMP-activated protein kinase pathway, the translocation of sterol regulatory element-binding protein-1 into the nucleus and forkhead box O1 into the cytoplasm was prevented. Alpha-lipoic acid increased adipose triacylglycerol lipase expression and decreased fatty acid synthase abundance. In in vivo and in vitro studies, alpha-lipoic acid also increased nuclear NF-E2-related factor 2 levels and downstream target amounts via the sirtuin 1 pathway. Alpha-lipoic acid eventually reduced intrahepatic and serum triglyceride content. The protective effects of alpha-lipoic acid on hepatic steatosis appear to be associated with the transcription factors sterol regulatory element-binding protein-1, forkhead box O1 and NF-E2-related factor 2. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
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Evert, M; Frau, M; Tomasi, M L; Latte, G; Simile, M M; Seddaiu, M A; Zimmermann, A; Ladu, S; Staniscia, T; Brozzetti, S; Solinas, G; Dombrowski, F; Feo, F; Pascale, R M; Calvisi, D F
2013-11-12
The DNA-repair gene DNA-dependent kinase catalytic subunit (DNA-PKcs) favours or inhibits carcinogenesis, depending on the cancer type. Its role in human hepatocellular carcinoma (HCC) is unknown. DNA-dependent protein kinase catalytic subunit, H2A histone family member X (H2AFX) and heat shock transcription factor-1 (HSF1) levels were assessed by immunohistochemistry and/or immunoblotting and qRT-PCR in a collection of human HCC. Rates of proliferation, apoptosis, microvessel density and genomic instability were also determined. Heat shock factor-1 cDNA or DNA-PKcs-specific siRNA were used to explore the role of both genes in HCC. Activator protein 1 (AP-1) binding to DNA-PKcs promoter was evaluated by chromatin immunoprecipitation. Kaplan-Meier curves and multivariate Cox model were used to study the impact on clinical outcome. Total and phosphorylated DNA-PKcs and H2AFX were upregulated in HCC. Activated DNA-PKcs positively correlated with HCC proliferation, genomic instability and microvessel density, and negatively with apoptosis and patient's survival. Proliferation decline and massive apoptosis followed DNA-PKcs silencing in HCC cell lines. Total and phosphorylated HSF1 protein, mRNA and activity were upregulated in HCC. Mechanistically, we demonstrated that HSF1 induces DNA-PKcs upregulation through the activation of the MAPK/JNK/AP-1 axis. DNA-dependent protein kinase catalytic subunit transduces HSF1 effects in HCC cells, and might represent a novel target and prognostic factor in human HCC.
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Lim, Y P; Low, B C; Lim, J; Wong, E S; Guy, G R
1999-07-02
FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.
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A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.
Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J
2017-09-19
Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
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Role of Matricellular Proteins in Disorders of the Central Nervous System.
Jayakumar, A R; Apeksha, A; Norenberg, M D
2017-03-01
Matricellular proteins (MCPs) are actively expressed non-structural proteins present in the extracellular matrix, which rapidly turnover and possess regulatory roles, as well as mediate cell-cell interactions. MCPs characteristically contain binding sites for other extracellular proteins, cell surface receptors, growth factors, cytokines and proteases, that provide structural support for surrounding cells. MCPs are present in most organs, including brain, and play a major role in cell-cell interactions and tissue repair. Among the MCPs found in brain include thrombospondin-1/2, secreted protein acidic and rich in cysteine family (SPARC), including Hevin/SC1, Tenascin C and CYR61/Connective Tissue Growth Factor/Nov family of proteins, glypicans, galectins, plasminogen activator inhibitor (PAI-1), autotaxin, fibulin and perisostin. This review summarizes the potential role of MCPs in the pathogenesis of major neurological disorders, including Alzheimer's disease, amyotrophic lateral sclerosis, ischemia, trauma, hepatic encephalopathy, Down's syndrome, autism, multiple sclerosis, brain neoplasms, Parkinson's disease and epilepsy. Potential therapeutic opportunities of MCP's for these disorders are also considered in this review.
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Aranha, C; Natraj, U; Iyer, K S; Shahani, S
1998-03-01
Our purpose was to determine whether lymphocytes synthesize proteins during pregnancy, to observe whether one of the proteins synthesized has early pregnancy factor (EPF)-like activity and to isolate and purify this molecule from culture supernatants obtained from stimulated lymphocytes of pregnant women. Lymphocyte proliferation assay and 35S-methionine labeling were done to study de novo synthesis of proteins followed by autoradiography to confirm synthesis of proteins. The rosette inhibition assay was used for detection of the EPF-like molecule. Gel filtration on Sephadex G-100 and RPHPLC were used for purification of the EPF-like molecule. The rate of incorporation of 35S-methionine was significantly higher in the lymphocytes of pregnant women compared to those of the control, and autoradiography confirmed the synthesis of proteins during pregnancy. There is a total protein enhancement trend observed during the first trimester that declines toward term. The EPF-like molecule is observed to be synthesized during all the trimesters of pregnancy. This molecule, when purified, showed a single homogeneous biologically active peak. The results indicated that there is an enhancement of existing protein or synthesis of new proteins during pregnancy. The EPF-like molecule is one of the many proteins synthesized and secreted by lymphocytes during pregnancy that, when purified, is biologically active.
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Shepard, A R; Zhang, W; Eberhardt, N L
1994-01-21
We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.
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Inhibition of Retinoblastoma Protein Inactivation
2016-09-01
Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY CLASSIFICATION OF: 17...developed a method to perform fragment based screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor...cancer, cell-cycle inhibition, activation, modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography
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Al-Khouri, Anna Maria; Paule, Marvin R.
2002-01-01
In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852
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Al-Khouri, Anna Maria; Paule, Marvin R
2002-02-01
In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.
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Gauldie, J; Richards, C; Harnish, D; Lansdorp, P; Baumann, H
1987-01-01
One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Images PMID:2444978
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Role of hypoxia-inducible factor-{alpha} in hepatitis-B-virus X protein-mediated MDR1 activation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa
2007-06-01
The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and induced the nuclear translocation of C/EBP{beta}. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1{alpha} siRNAmore » but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1{alpha} activation, and suggest HIF-1{alpha} for the therapeutic target of HBV-mediated chemoresistance.« less
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NASA Astrophysics Data System (ADS)
Li, Q. Q.; Xu, R.; Hunt, A. G.; Falcone, D. L.
Plants are constantly challenged by numerous environmental stresses both biotic and abiotic It is clear that plants have evolved to counter these stresses using all but limited means We recently discovered the potential role of a messenger RNA processing factor namely the Arabidopsis cleavage and polyadenylation specificity factor 30 kDa subunit AtCPSF30 when a mutant deficient in this factor displayed altered responses to an array of abiotic stresses This AtCPSF30 mutant named oxt6 exhibited an elevated tolerance to oxidative stress Microarray experiments of oxt6 and its complemented lines revealed an altered gene expression profile among which were antioxidative defense genes Interestingly the same gene encoding AtCPSF30 can also be transcribed into a large transcript that codes for a potential splicing factor Both protein products have a domain for RNA binding and a calmodulin binding domain activities of which have been confirmed by biochemical assays Surprisingly binding of AtCPSF30 to calmodulin inhibits the RNA-binding activity of the protein Mutational analysis shows that a small part of the protein is responsible for calmodulin binding and point mutations in this region abolished both RNA binding activity and the inhibition of this activity by calmodulin Analyses of the potential splicing factor are on going and the results will be presented The interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through
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Effects on coagulation factor production following primary hepatomitogen-induced direct hyperplasia.
Tatsumi, Kohei; Ohashi, Kazuo; Taminishi, Sanae; Takagi, Soichi; Utoh, Rie; Yoshioka, Akira; Shima, Midori; Okano, Teruo
2009-11-14
To investigate the molecular mechanisms involved in coagulation factor expression and/or function during direct hyperplasia (DH)-mediated liver regeneration. Direct hyperplasia-mediated liver regeneration was induced in female C57BL/6 mice by administering 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a representative hepatomitogen. Mice were weighed and sacrificed at various time points [Day 0 (D0: prior to injection), 3 h, D1, D2, D3, and D10] after TCPOBOP administration to obtain liver and blood samples. Using the RNA samples extracted from the liver, a comprehensive analysis was performed on the hepatic gene expression profiling of coagulation-related factors by real-time RT-PCR (fibrinogen, prothrombin, factors V, VII, VIII, IX, X, XI, XII, XIIIbeta, plasminogen, antithrombin, protein C, protein S, ADAMTS13, and VWF). The corresponding plasma levels of coagulation factors (fibrinogen, prothrombin, factors V, VII, VIII, IX, X, XI, XII, XIII, and VWF) were also analyzed and compared with their mRNA levels. Gavage administration of TCPOBOP (3 mg/kg body weight) resulted in a marked and gradual increase in the weight of the mouse livers relative to the total body weight to 220% by D10 relative to the D0 (control) ratios. At the peak of liver regeneration (D1 and D2), the gene expression levels for most of the coagulation-related factors (fibrinogen, prothrombin, factors V, VII, VIII, IX, XI, XII, XIIIbeta, plasminogen, antithrombin, protein C, ADAMTS13, VWF) were found to be down-regulated in a time-dependent manner, and gradually recovered by D10 to the basal levels. Only mRNA levels of factor X and protein S failed to show any decrease during the regenerative phase. As for the plasma levels, 5 clotting factors (prothrombin, factors VIII, IX, XI, and XII) demonstrated a significant decrease (P<0.05) during the regeneration phase compared with D0. Among these 5 factors, factor IX and factor XI showed the most dramatic decline in their activities by about 50% at D2 compared to the basal levels, and these reductions in plasma activity for both factors were consistent with our RT-PCR findings. In contrast, the plasma activities of the other coagulation factors (fibrinogen, factors V, VII, XIII, and VWF) were not significantly reduced, despite the reduction in the liver mRNA levels. Unlike the other factors, FX showed a temporal increase in its plasma activity, with significant increases (P<0.05) detected at D1. Investigating the coagulation cascade protein profiles during liver regeneration by DH may help to better understand the basic biology of the liver under normal and pathological conditions.
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Sakai, Eiko; Aoki, Yuri; Yoshimatsu, Masako; Nishishita, Kazuhisa; Iwatake, Mayumi; Fukuma, Yutaka; Okamoto, Kuniaki; Tanaka, Takashi; Tsukuba, Takayuki
2016-07-15
Osteoclasts are multinucleated bone-resorbing cells that differentiate in response to receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). Enhanced osteoclastogenesis contributes to bone diseases, such as osteoporosis and rheumatoid arthritis. Rubus parvifolius L. is traditionally used as an herbal medicine for rheumatism; however, its detailed chemical composition and the molecular mechanisms responsible for its biological action have not been elucidated. To investigate the mechanisms by which R. parvifolius L. extract and its major constituent sanguiin H-6, inhibit osteoclastogenesis and bone resorption. Cell proliferation, cell differentiation, and bone resorption were detected in vitro. Inhibition of signaling pathways, marker protein expression, and protein nuclear translocation were evaluated by western blot analysis. Tumor necrosis factor-α (TNF-α)-mediated osteoclastogenesis was examined in vivo. R. parvifolius L. extract inhibited the bone-resorption activity of osteoclasts. In addition, sanguiin H-6 markedly inhibited RANKL-induced osteoclast differentiation and bone resorption, reduced reactive oxygen species production, and inhibited the phosphorylation of inhibitor of NF-κB alpha (IκBα) and p38 mitogen-activated protein kinase. Sanguiin H-6 also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), cathepsin K, and c-Src. Moreover, sanguiin H-6 inhibited the nuclear translocation of NFATc1, c-Fos, and NF-κB in vitro, as well as TNF-α-mediated osteoclastogenesis in vivo. Our data revealed that R. parvifolius L. has anti-bone resorption activity and suggest that its constituent, sanguiin H-6, can potentially be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and subsequent bone destruction. Copyright © 2016 Elsevier GmbH. All rights reserved.
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α-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes
Potapovich, AI; Pastore, S; Kostyuk, VA; Lulli, D; Mariani, V; De Luca, C; Dudich, EI; Korkina, LG
2009-01-01
Background and purpose: The immunomodulatory effects of α-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. Experimental approach: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFκB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFκB DNA binding activity was measured by specific assays. Nitric oxide and H2O2 production and redox status were assessed by fluorescent probe and biochemical methods. Key results: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H2O2 and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFκB was activated by AFP alone or by its combination with UVA. Conclusions and implications: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin. PMID:19785658
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Factors Affecting EWS-FLI1 Activity in Ewing's Sarcoma
Herrero-Martin, David; Fourtouna, Argyro; Niedan, Stephan; Riedmann, Lucia T.; Schwentner, Raphaela; Aryee, Dave N. T.
2011-01-01
Ewing's sarcoma family tumors (ESFT) are characterized by specific chromosomal translocations, which give rise to EWS-ETS chimeric proteins. These aberrant transcription factors are the main pathogenic drivers of ESFT. Elucidation of the factors influencing EWS-ETS expression and/or activity will guide the development of novel therapeutic agents against this fatal disease. PMID:22135504
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Post-translational regulation of WRKY transcription factors in plant immunity.
Ishihama, Nobuaki; Yoshioka, Hirofumi
2012-08-01
Plants have evolved immune system to protect themselves against invading pathogens. Recent research has illustrated that signaling networks, after perception of diverse pathogen-derived signals, facilitate transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. WRKY proteins, which comprise a large family of plant transcription factors, are key players in plant immune responses. WRKY transcription factors participate in the control of defense-related genes either as positive or as negative regulators, and essentially are regulated at the transcriptional level. Emerging evidence emphasizes that group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are also activated by MAPK-dependent phosphorylation, underlining their importance in plant immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.
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DiGiacomo, Vincent; Marivin, Arthur; Garcia-Marcos, Mikel
2018-01-23
Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the "Gα-binding and -activating (GBA) motif". So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subunits in vitro and activates G protein signaling in cells. The importance of this mechanism of signal transduction is highlighted by the fact that its dysregulation underlies human diseases, such as cancer, which has made the proteins attractive new candidates for therapeutic intervention. Here we discuss recent discoveries on the structural basis of GBA-mediated activation of G proteins and its evolutionary conservation and compare them with the better-studied mechanism mediated by GPCRs.
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Gando, Satoshi; Mayumi, Toshihiko; Ukai, Tomohiko
2018-01-01
The pathophysiological mechanisms of acute coagulopathy of trauma-shock (ACOTS) are reported to include activated protein C-mediated suppression of thrombin generation via the proteolytic inactivation of activated Factor V (FVa) and FVIIIa; an increased fibrinolysis via neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C. The aims of this study are to review the evidences for the role of activated protein C in thrombin generation and fibrinolysis and to validate the diagnosis of ACOTS based on the activated protein C dynamics. We conducted systematic literature search (2007-2017) using PubMed, the Cochrane Database of Systematic Reviews (CDSR), and the Cochrane Central Register of Controlled Trials (CENTRAL). Clinical studies on trauma that measured activated protein C or the circulating levels of activated protein C-related coagulation and fibrinolysis markers were included in our study. Out of 7613 studies, 17 clinical studies met the inclusion criteria. The levels of activated protein C in ACOTS were inconsistently decreased, showed no change, or were increased in comparison to the control groups. Irrespective of the activated protein C levels, thrombin generation was always preserved or highly elevated. There was no report on the activated protein C-mediated neutralization of PAI-1 with increased fibrinolysis. No included studies used unified diagnostic criteria to diagnose ACOTS and those studies also used different terms to refer to the condition known as ACOTS. None of the studies showed direct cause and effect relationships between activated protein C and the suppression of coagulation and increased fibrinolysis. No definitive diagnostic criteria or unified terminology have been established for ACOTS based on the activated protein C dynamics.
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Complement factor H-related proteins in IgA nephropathy-sometimes a gentle nudge does the trick.
Thurman, Joshua M; Laskowski, Jennifer
2017-10-01
Complement activation probably contributes to glomerular inflammation and damage in IgA nephropathy. In this issue, 2 groups report that levels of factor H-related protein 1 are elevated in patients with IgA nephropathy and correlate with disease progression. These studies provide new evidence that the complement cascade is important to the pathogenesis of this disease. These results also suggest that factor H-related protein 1 levels may be useful for identifying those patients at high risk of disease progression. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
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Jutooru, Indira; Chadalapaka, Gayathri; Lei, Ping; Safe, Stephen
2010-01-01
Curcumin activates diverse anticancer activities that lead to inhibition of cancer cell and tumor growth, induction of apoptosis, and antiangiogenic responses. In this study, we observed that curcumin inhibits Panc28 and L3.6pL pancreatic cancer cell and tumor growth in nude mice bearing L3.6pL cells as xenografts. In addition, curcumin decreased expression of p50 and p65 proteins and NFκB-dependent transactivation and also decreased Sp1, Sp3, and Sp4 transcription factors that are overexpressed in pancreatic cancer cells. Because both Sp transcription factors and NFκB regulate several common genes such as cyclin D1, survivin, and vascular endothelial growth factor that contribute to the cancer phenotype, we also investigated interactions between Sp and NFκB transcription factors. Results of Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate that both p50 and p65 are Sp-regulated genes and that inhibition of constitutive or tumor necrosis factor-induced NFκB by curcumin is dependent on down-regulation of Sp1, Sp3, and Sp4 proteins by this compound. Curcumin also decreased mitochondrial membrane potential and induced reactive oxygen species in pancreatic cancer cells, and this pathway is required for down-regulation of Sp proteins in these cells, demonstrating that the mitochondriotoxic effects of curcumin are important for its anticancer activities. PMID:20538607
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Broghammer, Angelique; Krusell, Lene; Blaise, Mickaël; Sauer, Jørgen; Sullivan, John T.; Maolanon, Nicolai; Vinther, Maria; Lorentzen, Andrea; Madsen, Esben B.; Jensen, Knud J.; Roepstorff, Peter; Thirup, Søren; Ronson, Clive W.; Thygesen, Mikkel B.; Stougaard, Jens
2012-01-01
Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana. The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man3XylFucGlcNAc4, were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor–ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The Kd values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes. PMID:22859506
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NASA Technical Reports Server (NTRS)
McCarthy, T. L.; Ji, C.; Chen, Y.; Kim, K.; Centrella, M.
2000-01-01
Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-delta (C/EBPdelta) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBPdelta and C/EBPbeta expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBPdelta and C/EBPbeta on IGF-I expression by osteoblasts.
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Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A
2013-03-01
The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via different subsignaling pathways. Analyses of the expression levels of dozens of genes and the protein-protein interactions among them demonstrated that CD and UC have relatively similar gene expression signatures, whereas the gene expression signatures of T1D and JRA relatively differ from the signatures of the other autoimmune diseases. These diseases are the only ones activated via the Fcɛ pathway. The relevant genes and pathways reported in this study are discussed at length, and may be helpful in the diagnoses and understanding of autoimmunity and/or specific autoimmune diseases.
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... the blood to trigger the activity of the enzyme thrombin. Active thrombin then forms long protein strands that clump together with platelets and red blood cells to form clots. Read less Risk Factors Risk factors for VTE include a history of a previous VTE event; surgery; medical conditions ...
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Lee, Samuel; Min Kim, Soo; Dotimas, James; Li, Letitia; Feener, Edward P; Baldus, Stephan; Myers, Ronald B; Chutkow, William A; Patwari, Parth; Yoshioka, Jun; Lee, Richard T
2014-06-01
The endoplasmic reticulum (ER) is responsible for protein folding, modification, and trafficking. Accumulation of unfolded or misfolded proteins represents the condition of ER stress and triggers the unfolded protein response (UPR), a key mechanism linking supply of excess nutrients to insulin resistance and type 2 diabetes in obesity. The ER harbors proteins that participate in protein folding including protein disulfide isomerases (PDIs). Changes in PDI activity are associated with protein misfolding and ER stress. Here, we show that thioredoxin-interacting protein (Txnip), a member of the arrestin protein superfamily and one of the most strongly induced proteins in diabetic patients, regulates PDI activity and UPR signaling. We found that Txnip binds to PDIs and increases their enzymatic activity. Genetic deletion of Txnip in cells and mice led to increased protein ubiquitination and splicing of the UPR regulated transcription factor X-box-binding protein 1 (Xbp1s) at baseline as well as under ER stress. Our results reveal Txnip as a novel direct regulator of PDI activity and a feedback mechanism of UPR signaling to decrease ER stress. © 2014 Brigham and Women's Hospital. Published under the terms of the CC BY 4.0 license.
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Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice.
Overman, Jeroen; Fontaine, Frank; Moustaqil, Mehdi; Mittal, Deepak; Sierecki, Emma; Sacilotto, Natalia; Zuegg, Johannes; Robertson, Avril Ab; Holmes, Kelly; Salim, Angela A; Mamidyala, Sreeman; Butler, Mark S; Robinson, Ashley S; Lesieur, Emmanuelle; Johnston, Wayne; Alexandrov, Kirill; Black, Brian L; Hogan, Benjamin M; De Val, Sarah; Capon, Robert J; Carroll, Jason S; Bailey, Timothy L; Koopman, Peter; Jauch, Ralf; Smyth, Mark J; Cooper, Matthew A; Gambin, Yann; Francois, Mathias
2017-01-31
Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics.
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Ratman, Dariusz; Vanden Berghe, Wim; Dejager, Lien; Libert, Claude; Tavernier, Jan; Beck, Ilse M; De Bosscher, Karolien
2013-11-05
The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
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Bacteria-Phagocyte Interactions: Emerging Tactics in an Ancient Rivalry
1990-01-01
afhitan. mechanisms by which microbes cvade the deposi- Mimicry of decay -accelerating factor aExample. T ’ruzi tion of immunogiobulin and complement on...their , Possible Isis of decay accelerating factor on host cell, surfaces have been well-studied (Table 2). For Example. Bacterial phospholipase example...activators of protein that mimics the action of decay accelerat- the alternate complement pathway 1171. ing factor (DAF) [261. This protein is part of a
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DNA wrapping and distortion by an oligomeric homeodomain protein.
Williams, Hannah; Jayaraman, Padma-Sheela; Gaston, Kevin
2008-10-31
Many transcription factors alter DNA or chromatin structure. Changes in chromatin structure are often brought about by the recruitment of chromatin-binding proteins, chromatin-modifying proteins, or other transcription co-activator or co-repressor proteins. However, some transcription factors form oligomeric assemblies that may themselves induce changes in DNA conformation and chromatin structure. The proline-rich homeodomain (PRH/Hex) protein is a transcription factor that regulates cell differentiation and cell proliferation, and has multiple roles in embryonic development. Earlier, we showed that PRH can repress transcription by multiple mechanisms, including the recruitment of co-repressor proteins belonging to the TLE family of chromatin-binding proteins. Our in vivo crosslinking studies have shown that PRH forms oligomeric complexes in cells and a variety of biophysical techniques suggest that the protein forms octamers. However, as yet we have little knowledge of the role played by PRH oligomerisation in the regulation of promoter activity or of the architecture of promoters that are regulated directly by PRH in cells. Here, we compare the binding of PRH and the isolated PRH homeodomain to DNA fragments with single and multiple PRH sites, using gel retardation assays and DNase I and chemical footprinting. We show that the PRH oligomer binds to multiple sites within the human Goosecoid promoter with high affinity and that the binding of PRH brings about DNA distortion. We suggest that PRH octamers wrap DNA in order to bring about transcriptional repression.
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Vumbaca, Frank; Phoenix, Kathryn N.; Rodriguez-Pinto, Daniel; Han, David K.; Claffey, Kevin P.
2008-01-01
Vascular endothelial growth factor (VEGF) is a key angiogenic factor expressed under restricted nutrient and oxygen conditions in most solid tumors. The expression of VEGF under hypoxic conditions requires transcription through activated hypoxia-inducible factor 1 (HIF-1), increased mRNA stability, and facilitated translation. This study identified double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), a specific isoform of the DRBP family, as a VEGF mRNA-binding protein which plays a key role in VEGF mRNA stability and protein synthesis under hypoxia. The DRBP76/NF90 protein binds to a human VEGF 3′ untranslated mRNA stability element. RNA interference targeting the DRBP76/NF90 isoform limited hypoxia-inducible VEGF mRNA and protein expression with no change in HIF-1-dependent transcriptional activity. Stable repression of DRBP76/NF90 in MDA-MB-435 breast cancer cells demonstrated reduced polysome-associated VEGF mRNA levels under hypoxic conditions and reduced mRNA stability. Transient overexpression of the DRBP76/NF90 protein increased both VEGF mRNA and protein levels synthesized under normoxic and hypoxic conditions. Cells with stable repression of the DRBP76/NF90 isoform showed reduced tumorigenic and angiogenic potential in an orthotopic breast tumor model. These data demonstrate that the DRBP76/NF90 isoform facilitates VEGF expression by promoting VEGF mRNA loading onto polysomes and translation under hypoxic conditions, thus promoting breast cancer growth and angiogenesis in vivo. PMID:18039850
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Kim, Hyo Jung; Kim, Il Soon; Dong, Yin; Lee, Ik-Soo; Kim, Jin Sook; Kim, Jong-Sang; Woo, Je-Tae; Cha, Byung-Yoon
2015-04-20
The melanin-inducing properties of cirsimaritin were investigated in murine B16F10 cells. Cirsimaritin is an active flavone with methoxy groups, which is isolated from the branches of Lithocarpus dealbatus. Tyrosinase activity and melanin content in murine B16F10 melanoma cells were increased by cirsimaritin in a dose-dependent manner. Western blot analysis revealed that tyrosinase, tyrosinase-related protein (TRP) 1, TRP2 protein levels were enhanced after treatment with cirsimaritin for 48 h. Cirsimaritin also upregulated the expression of microphthalmia-associated transcription factor (MITF) after 24 h of treatment. Furthermore, cirsimaritin induced phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in a dose-dependent manner after treatment for 15 min. The cirsimaritin-mediated increase of tyrosinase activity was significantly attenuated by H89, a cAMP-dependent protein kinase A inhibitor. These findings indicate that cirsimaritin stimulates melanogenesis in B16F10 cells by activation of CREB as well as upregulation of MITF and tyrosinase expression, which was activated by cAMP signaling. Finally, the melanogenic effect of cirsimaritin was confirmed in human epidermal melanocytes. These results support the putative application of cirsimaritin in ultraviolet photoprotection and hair coloration treatments.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Barbon, Silvia, E-mail: silvia.barbon@yahoo.it
In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in themore » future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6 h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury. - Highlights: • TAT-CNTF is an optimized fusion protein that preserves neurotrophic activity. • In neural cell lines, TAT-CNTF triggers the activation of signal transduction. • Fast cellular uptake of TAT-CNTF was accomplished after cell treatment. • TAT-CNTF can be efficiently loaded on oxidized PVA cylinders for local delivery. • TAT-CNTF features make it ideal for peripheral nerve regeneration therapies.« less
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The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.
Chepelev, Nikolai L; Bennitz, Joshua D; Huang, Ting; McBride, Skye; Willmore, William G
2011-01-01
Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status. © 2011 Chepelev et al.
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Gan, Li; Ma, Delin; Li, Min; Yang, Fu-Chen; Rogers, Robert S; Wheatley, Joshua L; Koch, Lauren G; Britton, Steven L; Thyfault, John P; Geiger, Paige C; Stanford, John A
2018-05-01
Aerobic capacity is a strong predictor of mortality. Low capacity runner (LCR) rats exhibit reduced mitochondrial function in peripheral organs. A high fat diet (HFD) can worsen metabolic phenotype in LCR rats. Little is known about metabolic changes in the brains of these rats, however. This study examined protein markers of mitochondrial function and metabolism as a function of aerobic running capacity and an acute HFD in four brain regions: the striatum, hippocampus, hypothalamus, and substantia nigra. After 3 days HFD or chow diets, we measured peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1-α), nuclear respiratory factors 1 (Nrf-1), mitochondrial transcription factor A (TFAM), and phosphorylated (activated) AMP-activated protein kinase (p-AMPK) protein levels in the four brain regions. LCR rats exhibited lower levels of mitochondrial proteins (PGC1-α, Nrf-1, TFAM), and greater p-AMPK, in striatum, but not in the other brain regions. Mitochondrial protein levels were greater in HFD LCR striatum, while p-AMPK was lower in this group. Markers of lower mitochondrial biogenesis and increased metabolic demand were limited to the LCR striatum, which nevertheless maintained the capacity to respond to an acute HFD challenge. Copyright © 2018 Elsevier B.V. All rights reserved.
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Ji, Liang; Nazarali, Adil J.; Paterson, Phyllis G.
2013-01-01
Protein–energy malnutrition (PEM) exacerbates functional impairment caused by brain ischemia. This is correlated with reactive gliosis, which suggests an increased inflammatory response. The objective of the current study was to investigate if PEM increases hippocampal activation of nuclear factor κB (NFκB), a transcription factor that amplifies the inflammatory response involved in ischemic brain injury. Mongolian gerbils (11–12 weeks old) were randomly assigned to control diet (12.5% protein) or protein-deficient diet (2%) for 4 weeks. The 2% protein group had a 15% decrease in voluntary food intake (P<.001; unpaired t test), resulting in PEM. Body weight after 4 weeks was 20% lower in the PEM group (P<.001). Gerbils were then exposed to sham surgery or global ischemia induced by 5-min bilateral common carotid artery occlusion. PEM independently increased hippocampal NFκB activation detected by electrophoretic mobility shift assay at 6 h after surgery (P=.014; 2-factor ANOVA). Ischemia did not significantly affect NFκB activation nor was there interaction between diet and ischemia. Serum glucose and cortisol concentrations at 6 h postischemia were unaltered by diet or ischemia. A second experiment using gerbils of the same age and feeding paradigm demonstrated that PEM also increases hippocampal NFκB activation in the absence of surgery. These findings suggest that PEM, which exists in 16% of elderly patients at admission for stroke, may worsen outcome by increasing activation of NFκB. Since PEM increased NFκB activation independent of ischemia or surgery, the data also have implications for the inflammatory response of the many individuals affected globally by PEM. PMID:18430555
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Kalra, Dinesh; Sivasubramanian, Natarajan; Mann, Douglas L
2002-05-07
Previous studies suggest that angiotensin II (Ang II) upregulates the expression of tumor necrosis factor (TNF) in nonmyocyte cell types; however, the effect of Ang II on TNF expression in the adult mammalian heart is not known. To determine whether Ang II was sufficient to provoke TNF biosynthesis in the adult heart, we examined the effects of Ang II in isolated buffer-perfused Langendorff feline hearts. Ang II (10(-7) mol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF mRNA and protein biosynthesis in the heart as well as in cultured adult cardiac myocytes. The effects of Ang II on myocardial TNF mRNA and protein synthesis were mediated through the angiotensin type 1 receptor (AT1R), insofar as an AT1R antagonist (AT1a) blocked the effects of Ang II, whereas an angiotensin type 2 receptor (AT2R) antagonist (AT2a) had no effect. Stimulation with Ang II led to the activation of nuclear factor-kappaB and activator protein-1 (AP-1), two transcription factors that are important for TNF gene expression. Nuclear factor-kappaB activation was accompanied by phosphorylation of IkappaBalpha on serine 32 as well as degradation of IkappaBalpha, suggesting that the effects of Ang II were mediated through an IkappaBalpha-dependent pathway. The important role of protein kinase C (PKC) was suggested by studies in which a phorbol ester triggered TNF biosynthesis, and a PKC inhibitor abrogated Ang II-induced TNF biosynthesis. These studies suggest that Ang II provokes TNF biosynthesis in the adult mammalian heart through a PKC-dependent pathway.
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De Santa Barbara, P; Bonneaud, N; Boizet, B; Desclozeaux, M; Moniot, B; Sudbeck, P; Scherer, G; Poulat, F; Berta, P
1998-11-01
For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.
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De Santa Barbara, Pascal; Bonneaud, Nathalie; Boizet, Brigitte; Desclozeaux, Marion; Moniot, Brigitte; Sudbeck, Peter; Scherer, Gerd; Poulat, Francis; Berta, Philippe
1998-01-01
For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis. PMID:9774680
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Beregovyi, S M; Chervinska, T M; Dranitsina, A S; Szabo, S; Tolstanova, G M
2015-01-01
Changes in redox status of gastric mucosa cells are the main pathogenic factor of gastric erosion and gastric ulcer development. Pro-oxidants can affect cell transcription activity via changes in redox-sensitive transcription factors. Egr-1 and Sp-1 may regulate the transcription of genes that are associated with the pathogenesis of gastric ulcer (growthfactors, cell cycle regulators, etc.). The aim of the present study was to reveal the possible involvement of zinc-finger transcriptionfactors Egr-1 & Sp-1 in the molecular mechanisms underlying gastric lesions caused by aspirin administration and stress. Gastric ulcer was induced in male rats (180-220 g) by immobilization stress combined with water-immersion (IMO-WI) or aspirin gavage (10 mg/100 g). The rats were euthanized 20 min, 1 hour, or 3 hours following the ulcerogenic factor exposure. Protein expression was determined by Western blot analysis and RT-PCR; levels of SH-groups of proteins were determined by method of Ellman et al. Development of gastric ulcer lesions was associated with twofold (P < 0.05) decrease in concentration of protein SH-groups in the rat gastric mucosa. These changes were accompanied by significant (P < 0.05) increase in the expression of Egr-1 mRNA and protein in both gastric ulcer models, and the changes in IMO-WI were more profound. Increased levels of Egr-1 were associated with the decrease in SpI protein levels. We showed for the first time the competitive interaction between redox-sensitive transcription factors Egr-1 and Sp1 in the early phases of gastric ulcer development, which might facilitate inducible transcriptional activity of Egr-1 at the expense of reduction in Sp1 activity.
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Liu, Jie; Li, Fanfan; Shu, Kuangyi; Chen, Tao; Wang, Xiaoou; Xie, Yaoqi; Li, Shanshan; Zhang, Zhaohua; Jin, Susu; Jiang, Minghua
2018-05-13
To investigate the effect of C-reactive protein on the activated partial thromboplastin time (APTT) (different activators) in different detecting systems. The C-reactive protein and coagulation test of 112 patients with the infectious disease were determined by automation protein analyzer IMMAG 800 and automation coagulation analyzer STA-R Evolution, respectively. The pooled plasma APTT with different concentrations of C-reactive protein was measured by different detecting system: STA-R Evolution (activator: silica, kaolin), Sysmex CS-2000i (activator: ellagic acid), and ACL TOP 700 (activator: colloidal silica). In addition, the self-made platelet lysate (phospholipid) was added to correct the APTT prolonged by C-reactive protein (150 mg/L) on STA-R Evolution (activator: silica) system. The good correlation between C-reactive protein and APTT was found on the STA-R Evolution (activator: silica) system. The APTT on the STA-R Evolution (activator: silica) system was prolonged by 24.6 second, along with increasing C-reactive protein concentration. And the APTT of plasma containing 150 mg/L C-reactive protein was shortened by 3.4-6.9 second when the plasma was mixed with self-made platelet lysate. However, the APTT was prolonged unobviously on other detecting systems including STA-R Evolution (activator: kaolin), Sysmex CS-2000i, and ACL TOP 700. C-reactive protein interferes with the detection of APTT, especially in STA-R Evolution (activator: silica) system. The increasing in C-reactive protein results in a false prolongation of the APTT (activator: silica), and it is most likely that C-reactive protein interferes the coagulable factor binding of phospholipid. © 2018 Wiley Periodicals, Inc.
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Dong, Ruizeng; Guo, Jianmin; Zhang, Zewei; Zhou, Yimin; Hua, Yonghong
2018-03-18
The aim of this study was to identify the anti-cancer mechanism of Polyphyllin I (PPI) on gastric cancer cells via its activity on cancer-associated fibroblasts (CAFs). We cultured purified gastric CAFs obtained from fresh human gastric cancer tissue and examined the effect of Polyphyllin I on CAF proliferation using a colorimetric viability assay. In addition, we established a nude mouse xenograft model to examine the effect of Polyphyllin I administration on tumorigenesis. Using Western analysis, we quantified protein expression of the CAF-derived cytokines fibroblast activation protein alpha (FAP), secreted protein acidic and cysteine rich (SPARC), stromal cell-derived factor 1 (SDF-1), hepatocyte growth factor tenascin-C (TNC), and hepatocyte growth factor (HGF) in both in vitro and in vivo models. We found that Polyphyllin I inhibits the proliferation of CAFs in a concentration-dependent manner. Following treatment with 2 μg/ml PPI for 24 h in vitro, the expression of FAP, SDF-1 and HGF protein in CAFs was significantly lower than that in the control group, but there was no significant difference in SPARC and TNC protein expression between the two groups. In the nude mouse xenograft model, the tumor inhibition rate was 45.5% when PPI was administered early and 29.4% with administration in the third week. The expression of FAP and HGF in the xenografts was significantly decreased, while the expression of SPARC, SDF-1, and TNC was largely unaltered. Altogether, these data suggest that Polyphyllin I can inhibit the proliferation of gastric cancer cells by downregulating the expression of FAP and HGF in CAFs in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.
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Osada, Eriko; Akematsu, Takahiko; Asano, Tomoya; Endoh, Hiroshi
2014-03-01
Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.
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Kimura, Koji; Inoue, Kengo; Okubo, Jun; Ueda, Yumiko; Kawaguchi, Kosuke; Sakurai, Hiroaki; Wada, Ikuo; Morita, Masashi; Imanaka, Tsuneo
2015-01-01
Newly synthesized secretory proteins are folded and assembled in the endoplasmic reticulum (ER), where an efficient protein quality control system performs a critically important function. When unfolded or aggregated proteins accumulate in the ER, certain signaling pathways such as the unfolded protein response (UPR) and ER-overload response (EOR) are functionally active in maintaining cell homeostasis. Recently we prepared Chinese hamster ovary (CHO) cells expressing mutant antithrombin (AT)(C95R) under control of the Tet-On system and showed that AT(C95R) accumulated in Russell bodies (RB), large distinctive structures derived from the ER. To characterize whether ER stress takes place in CHO cells, we examined characteristic UPR and EOR in ER stress responses. We found that the induction of ER chaperones such as Grp97, Grp78 and protein disulfide isomerase (PDI) was limited to a maximum of approximately two-fold. The processing of X-box-binding protein-1 (XBP1) mRNA and the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) subunit were not induced. Furthermore, the activation of nuclear factor-kappa B (NF-κB) was not observed. In contrast, CHO cells displayed UPR and EOR when the cells were treated with thapsigargin and tumor necrosis factor (TNF)-α, respectively. In addition, a portion of the mutant AT(C95R) was degraded through proteasomes and autophagy. CHO cells do respond to ER stress but the folding state of mutant AT(C95R) does not appear to activate the ER stress signal pathway.
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Park, Chang-Jin; Caddell, Daniel F.; Ronald, Pamela C.
2012-01-01
Plants are continuously challenged by pathogens including viruses, bacteria, and fungi. The plant immune system recognizes invading pathogens and responds by activating an immune response. These responses occur rapidly and often involve post-translational modifications (PTMs) within the proteome. Protein phosphorylation is a common and intensively studied form of these PTMs and regulates many plant processes including plant growth, development, and immunity. Most well-characterized pattern recognition receptors (PRRs), including Xanthomonas resistance 21, flagellin sensitive 2, and elongation factor-Tu receptor, possess intrinsic protein kinase activity and regulate downstream signaling through phosphorylation events. Here, we focus on the phosphorylation events of plant PRRs that play important roles in the immune response. We also discuss the role of phosphorylation in regulating mitogen-associated protein kinase cascades and transcription factors in plant immune signaling. PMID:22876255
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Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.
Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P
1993-06-25
Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.
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Co-factors Required for TLR7- and TLR9- dependent Innate Immune Responses
Chiang, Chih-yuan; Engel, Alex; Opaluch, Amanda M.; Ramos, Irene; Maestre, Ana M.; Secundino, Ismael; De Jesus, Paul D.; Nguyen, Quy T.; Welch, Genevieve; Bonamy, Ghislain M.C.; Miraglia, Loren J.; Orth, Anthony P.; Nizet, Victor; Fernandez-Sesma, Ana; Zhou, Yingyao; Barton, Gregory M.; Chanda, Sumit K.
2012-01-01
SUMMARY Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent pro-inflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis we identify 190 co-factors required for TLR7- and TLR9-directed signaling responses. A set of co-factors were cross-profiled for their activities downstream of several immunoreceptors, and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection. PMID:22423970
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[Systemic and local mechanisms leading to cachexia in cancer].
Grabiec, Kamil; Burchert, Marta; Milewska, Marta; Błaszczyk, Maciej; Grzelkowska-Kowalczyk, Katarzyna
2013-12-31
Cachexia is a multifactorial syndrome of atrophy of skeletal muscle and adipose tissue, resulting in progressive loss of body weight associated with low quality of life and poor prognosis in cancer. Studies on experimental animal models and observations on patients have shown that the soluble factors secreted by tumor cells and tissues of the patient can participate in regulation of the wasting process. Cachexia is often accompanied by anorexia, which is caused by predominance of signals inhibiting appetite in the hypothalamus, such as release of proopiomelanocortin and anorexigenic action of proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α). Cachexia is also accompanied by extensive metabolic changes consisting of increase of resting energy expenditure and disturbance of carbohydrate, protein and lipid metabolism. Increased expression of protein uncoupling phosphorylation leads to increased thermogenesis in skeletal muscle. Tumor tissue hypoxia caused by its growth beyond blood vessels activates the transcription factor HIF-1, which results in increase in glycolysis, and leads to lactic acid accumulation and activation of the energy inefficient Cori cycle. Loss of fat tissue is caused by increase of lipolysis induced by lipid-mobilizing factor (LMF) and proinflammatory cytokines. Skeletal muscle wasting in cachexia is caused by a reduction of protein synthesis at the stage of initiation and elongation of translation and the simultaneous increase of protein degradation via ubiquitin-dependent and lysosomal pathways. The main mediators of skeletal muscle wasting in cancer are proteolysis-inducing factor (PIF), proinflammatory cytokines, and angiotensin II acting through increased levels of reactive oxygen species (ROS) and nuclear factor NF-κB activation, as well as glucocorticoid activated FOXO transcription factors and myostatin. Understanding of the complexity of the interaction of factors produced by the tumor and the patient's body may form the basis for the development of effective treatments for cachexia in cancer and other pathological conditions.
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Carter, Angela M.; Gutowski, Stephen; Sternweis, Paul C.
2014-01-01
The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA. PMID:24855647
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NASA Astrophysics Data System (ADS)
Fuseler, John W.; Merrill, Dana M.; Rogers, Jennifer A.; Grisham, Matthew B.; Wolf, Robert E.
2006-07-01
Nuclear factor kappa B (NF-[kappa]B) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-[kappa]Bs, which prevent entry into the nucleus. Following cellular stimulation, the I-[kappa]Bs are rapidly degraded, activating NF-[kappa]B. The active form of NF-[kappa]B rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-[kappa]B is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-[kappa]B (p65 subunit, p65-NF-[kappa]B) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-[kappa]B protein in the nucleus determined biochemically. The appearance of p65-NF-[kappa]B in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-[kappa]B can be reliably determined from IOD measurements of the immunofluorescent labeled protein.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Suzuki, Shigeki; Kulkarni, Ashok B., E-mail: ak40m@nih.gov
2010-07-30
Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-{beta} signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understandingmore » of the latent TGF-{beta} activation process. In this study, we have identified heat shock protein 90 {beta} (HSP90{beta}) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90{beta} into extracellular space which inhibits the activation of latent TGF-{beta}1, and that there is a subsequent decrease in cell proliferation. TGF-{beta}1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90{beta}. Thus, extracellular HSP90{beta} is a negative regulator for the activation of latent TGF-{beta}1 modulating TGF-{beta} signaling in the extracellular domain. -- Research highlights: {yields} Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex. {yields} This complex consists of latency-associated peptide (LAP) and the mature ligand. {yields} The release of the mature ligand from LAP is the first step in TGF-{beta} activation. {yields} We identified for the first time a novel mechanism for this activation process. {yields} Heat shock protein 90 {beta} is discovered as a negative regulator for this process.« less
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The Murine Factor H-Related Protein FHR-B Promotes Complement Activation.
Cserhalmi, Marcell; Csincsi, Ádám I; Mezei, Zoltán; Kopp, Anne; Hebecker, Mario; Uzonyi, Barbara; Józsi, Mihály
2017-01-01
Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.
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β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.
Salto, Rafael; Vílchez, Jose D; Girón, María D; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M
2015-01-01
β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.
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β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells
Girón, María D.; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M.
2015-01-01
β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth. PMID:26267903
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Regulatory elements involved in tax-mediated transactivation of the HTLV-I LTR.
Seeler, J S; Muchardt, C; Podar, M; Gaynor, R B
1993-10-01
HTLV-I is the etiologic agent of adult T-cell leukemia. In this study, we investigated the regulatory elements and cellular transcription factors which function in modulating HTLV-I gene expression in response to the viral transactivator protein, tax. Transfection experiments into Jurkat cells of a variety of site-directed mutants in the HTLV-1 LTR indicated that each of the three motifs A, B, and C within the 21-bp repeats, the binding sites for the Ets family of proteins, and the TATA box all influenced the degree of tax-mediated activation. Tax is also able to activate gene expression of other viral and cellular promoters. Tax activation of the IL-2 receptor and the HIV-1 LTR is mediated through NF-kappa B motifs. Interestingly, sequences in the 21-bp repeat B and C motifs contain significant homology with NF-kappa B regulatory elements. We demonstrated that an NF-kappa B binding protein, PRDII-BF1, but not the rel protein, bound to the B and C motifs in the 21-bp repeat. PRDII-BF1 was also able to stimulate activation of HTLV-I gene expression by tax. The role of the Ets proteins on modulating tax activation was also studied. Ets 1 but not Ets 2 was capable of increasing the degree of tax activation of the HTLV-I LTR. These results suggest that tax activates gene expression by either direct or indirect interaction with several cellular transcription factors that bind to the HTLV-I LTR.
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Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D
2005-01-01
Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.
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2011-01-01
Background The aims of this study were to assess the intake of animal, plant and food group-specific protein, and to investigate their associations with socio-economic and lifestyle-related factors in Flemish preschoolers. Methods Three-day estimated dietary records were collected from 661 preschoolers aged 2.5-6.5 y (338 boys and 323 girls). Multiple linear regression analysis was used to investigate the association between animal, plant, and food group-specific protein intake and socio-economic and lifestyle factors. Results Animal proteins (mean 38 g/d) were the main source of total protein (mean 56 g/d), while mean plant protein intake amounted to 18 g/d. The group of meat, poultry, fish and eggs was the main contributor (51%) to animal protein intake, followed by milk and milk products (35%). Bread and cereals (41%) contributed most to the plant protein intake, followed by low-nutritious, energy-dense foods (21%). With higher educated fathers and mothers as reference, respectively, preschoolers with lower secondary and secondary paternal education had lower animal, dairy-, and meat-derived protein intakes, and those with lower secondary and secondary maternal education consumed less plant, and bread and cereal-derived proteins. Compared to children with high physical activity levels, preschoolers with low and moderate physical activity had lower animal and plant protein intakes. Significantly higher potatoes and grains-, and fish- derived proteins were reported for children of smoking mothers and fathers, respectively, compared to those of non-smoking mothers and fathers. Conclusions The total protein intake of Flemish preschoolers was sufficient according to the recommendations of the Belgian Superior Health Council. Parental level of education and smoking status might play a role in the sources of children's dietary proteins. PMID:21943312
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Early Low Protein Diet Aggravates Unbalance between Antioxidant Enzymes Leading to Islet Dysfunction
Theys, Nicolas; Clippe, André; Bouckenooghe, Thomas; Reusens, Brigitte; Remacle, Claude
2009-01-01
Background Islets from adult rat possess weak antioxidant defense leading to unbalance between superoxide dismutase (SOD) and hydrogen peroxide-inactivating enzymatic activities, catalase (CAT) and glutathione peroxidase (GPX) rending them susceptible to oxidative stress. We have shown that this vulnerability is influenced by maternal diet during gestation and lactation. Methodology/Principal Findings The present study investigated if low antioxidant activity in islets is already observed at birth and if maternal protein restriction influences the development of islet antioxidant defenses. Rats were fed a control diet (C group) or a low protein diet during gestation (LP) or until weaning (LPT), after which offspring received the control diet. We found that antioxidant enzymatic activities varied with age. At birth and after weaning, normal islets possessed an efficient GPX activity. However, the antioxidant capacity decreased thereafter increasing the potential vulnerability to oxidative stress. Maternal protein malnutrition changed the antioxidant enzymatic activities in islets of the progeny. At 3 months, SOD activity was increased in LP and LPT islets with no concomitant activation of CAT and GPX. This unbalance could lead to higher hydrogen peroxide production, which may concur to oxidative stress causing defective insulin gene expression due to modification of critical factors that modulate the insulin promoter. We found indeed that insulin mRNA level was reduced in both groups of malnourished offspring compared to controls. Analyzing the expression of such critical factors, we found that c-Myc expression was strongly increased in islets from both protein-restricted groups compared to controls. Conclusion and Significance Modification in antioxidant activity by maternal low protein diet could predispose to pancreatic islet dysfunction later in life and provide new insights to define a molecular mechanism responsible for intrauterine programming of endocrine pancreas. PMID:19568427
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Factor XI and Contact Activation as Targets for Antithrombotic Therapy
Gailani, David; Bane, Charles E.; Gruber, Andras
2015-01-01
Summary The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa, or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and factor X. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally-induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on factor XII, the zymogen of a protease (factor XIIa) that initiates contact activation when blood is exposed to foreign surfaces; and factor XI, the zymogen of the protease factor XIa, which links contact activation to the thrombin generation mechanism. In the case of factor XI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of factor XI may be more effective than low-molecular-weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here we review data on the role of factor XI and factor XII in thrombosis, and results of pre-clinical and human trials for therapies targeting these proteins. PMID:25976012
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Marro, Samuele; Chiabrando, Deborah; Messana, Erika; Stolte, Jens; Turco, Emilia; Tolosano, Emanuela; Muckenthaler, Martina U
2010-08-01
Macrophages of the reticuloendothelial system play a key role in recycling iron from hemoglobin of senescent or damaged erythrocytes. Heme oxygenase 1 degrades the heme moiety and releases inorganic iron that is stored in ferritin or exported to the plasma via the iron export protein ferroportin. In the plasma, iron binds to transferrin and is made available for de novo red cell synthesis. The aim of this study was to gain insight into the regulatory mechanisms that control the transcriptional response of iron export protein ferroportin to hemoglobin in macrophages. Iron export protein ferroportin mRNA expression was analyzed in RAW264.7 mouse macrophages in response to hemoglobin, heme, ferric ammonium citrate or protoporphyrin treatment or to siRNA mediated knockdown or overexpression of Btb And Cnc Homology 1 or nuclear accumulation of Nuclear Factor Erythroid 2-like. Iron export protein ferroportin promoter activity was analyzed using reporter constructs that contain specific truncations of the iron export protein ferroportin promoter or mutations in a newly identified MARE/ARE element. We show that iron export protein ferroportin is transcriptionally co-regulated with heme oxygenase 1 by heme, a degradation product of hemoglobin. The protoporphyrin ring of heme is sufficient to increase iron export protein ferroportin transcriptional activity while the iron released from the heme moiety controls iron export protein ferroportin translation involving the IRE in the 5'untranslated region. Transcription of iron export protein ferroportin is inhibited by Btb and Cnc Homology 1 and activated by Nuclear Factor Erythroid 2-like involving a MARE/ARE element located at position -7007/-7016 of the iron export protein ferroportin promoter. This finding suggests that heme controls a macrophage iron recycling regulon involving Btb and Cnc Homology 1 and Nuclear Factor Erythroid 2-like to assure the coordinated degradation of heme by heme oxygenase 1, iron storage and detoxification by ferritin, and iron export by iron export protein ferroportin.
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Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.
Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.
2016-01-01
Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the activation domain of ‘free’ Tlo protein competes with DNA bound transcription factors for targets that regulate key aspects of C. albicans cell physiology. PMID:27741243
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Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.
Liu, Zhongle; Moran, Gary P; Sullivan, Derek J; MacCallum, Donna M; Myers, Lawrence C
2016-10-01
Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of 'free,' non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large 'free' pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the activation domain of 'free' Tlo protein competes with DNA bound transcription factors for targets that regulate key aspects of C. albicans cell physiology.
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Gupta, Indranil; Ganguly, Souradipta; Rozanas, Christine R; Stuehr, Dennis J; Panda, Koustubh
2016-07-19
Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage.
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Gupta, Indranil; Ganguly, Souradipta; Rozanas, Christine R.; Stuehr, Dennis J.
2016-01-01
Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage. PMID:27382160
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Hassoun, Shimaa M; Abdel-Rahman, Noha; Eladl, Entsar I; El-Shishtawy, Mamdouh M
2017-06-01
Angiogenesis plays important roles in progression of hepatocellular carcinoma. The antiangiogenic mechanisms of vitexicarpine are not fully defined. Therefore, we conducted the following study to evaluate the antiangiogenic mechanism and antitumor activity of vitexicarpine in vivo model of hepatocellular carcinoma through modulation of vascular endothelial growth factor signaling pathway. Hepatocellular carcinoma was induced in Sprague Dawley rats by thioacetamide. Hepatocellular carcinoma was assessed by measuring serum alpha-fetoprotein and investigating liver sections stained with hematoxylin/eosin. Hepatocellular carcinoma rats were injected with vitexicarpine (150 mg/kg) for 2 weeks. Hepatic vascular endothelial growth factor was measured by enzyme-linked immunosorbent assay. Protein and expression of hepatic phospho-Ser473-AKT (p-AKT) and phospho-Tyr419-Src (p-Src) were determined. The apoptotic pathway was evaluated by assessment of protein expression of caspase-3. Vitexicarpine increased rats' survival time and decreased serum alpha-fetoprotein as well as it ameliorated fibrosis and massive hepatic tissue breakdown. It attenuated hepatocellular carcinoma-induced protein and gene expression of vascular endothelial growth factor, p-AKT, p-Src, and caspase-3. In conclusion, this study suggests that vitexicarpine possesses both antiangiogenic and antitumor activities through inhibition of vascular endothelial growth factor, p-AKT/AKT, and p-Src with subsequent inhibition of apoptotic pathway.
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4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression
Shives, Katherine D.; Massey, Aaron R.; May, Nicholas A.; Morrison, Thomas E.; Beckham, J. David
2016-01-01
West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome. PMID:27763553
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Dysfunction of protein kinase FA/GSK-3 alpha in lymphocytes of patients with schizophrenic disorder.
Yang, S D; Yu, J S; Lee, T T; Yang, C C; Ni, M H; Yang, Y Y
1995-09-01
As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP.Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA/glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA/GSK-3 alpha. Immunoblotting and kinase activity analysis in an anti-kinase FA/GSK-3 alpha immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA/GSK-3 alpha in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA/GSK-3 alpha activity in the high levels of 14.8 +/- 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA/GSK-3 alpha activity in the low levels of 2.8 +/- 1.6 units/mg, indicating that the different levels of kinase FA/GSK-3 alpha activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide initial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA/GSK-3 alpha, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes.
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Tan, Y; Low, K G; Boccia, C; Grossman, J; Comb, M J
1994-01-01
Growth factors and cyclic AMP (cAMP) are known to activate distinct intracellular signaling pathways. Fibroblast growth factor (FGF) activates ras-dependent kinase cascades, resulting in the activation of MAP kinases, whereas cAMP activates protein kinase A. In this study, we report that growth factors and cAMP act synergistically to stimulate proenkephalin gene expression. Positive synergy between growth factor- and cAMP-activated signaling pathways on gene expression has not been previously reported, and we suggest that these synergistic interactions represent a useful model for analyzing interactions between these pathways. Transfection and mutational studies indicate that both FGF-dependent gene activation and cAMP-dependent gene activation require cAMP response element 2 (CRE-2), a previously characterized cAMP-dependent regulatory element. Furthermore, multiple copies of this element are sufficient to confer FGF regulation upon a minimal promoter, indicating that FGF and cAMP signaling converge upon transcription factors acting at CRE-2. Among many different ATF/AP-1 factors tested, two factors, ATF-3 and c-Jun, stimulate proenkephalin transcription in an FGF- or Ras-dependent fashion. Finally, we show that ATF-3 and c-Jun form heterodimeric complexes in SK-N-MC cells and that the levels of both proteins are increased in response to FGF but not cAMP. Together, these results indicate that growth factor- and cAMP-dependent signaling pathways converge at CRE-2 to synergistically stimulate gene expression and that ATF-3 and c-Jun regulate proenkephalin transcription in response to both growth factor- and cAMP-dependent intracellular signaling pathways. Images PMID:7935470
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2010-01-01
Background Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions. PMID:20122244
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2012-01-01
Background The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here. Findings Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs). Conclusions These findings add TBC1D20 to the network of host factors regulating HIV replication cycle. PMID:22260459
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Histone H1 functions as a stimulatory factor in backup pathways of NHEJ
Rosidi, Bustanur; Wang, Minli; Wu, Wenqi; Sharma, Aparna; Wang, Huichen; Iliakis, George
2008-01-01
DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ. PMID:18250087
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AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.
Carnegie, Graeme K; Soughayer, Joseph; Smith, F Donelson; Pedroja, Benjamin S; Zhang, Fang; Diviani, Dario; Bristow, Michael R; Kunkel, Maya T; Newton, Alexandra C; Langeberg, Lorene K; Scott, John D
2008-10-24
Elevated catecholamines in the heart evoke transcriptional activation of the Myocyte Enhancer Factor (MEF) pathway to induce a cellular response known as pathological myocardial hypertrophy. We have discovered that the A-Kinase Anchoring Protein (AKAP)-Lbc is upregulated in hypertrophic cardiomyocytes. It coordinates activation and movement of signaling proteins that initiate MEF2-mediated transcriptional reprogramming events. Live-cell imaging, fluorescent kinase activity reporters, and RNA interference techniques show that AKAP-Lbc couples activation of protein kinase D (PKD) with the phosphorylation-dependent nuclear export of the class II histone deacetylase HDAC5. These studies uncover a role for AKAP-Lbc in which increased expression of the anchoring protein selectively amplifies a signaling pathway that drives cardiac myocytes toward a pathophysiological outcome.
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Auto-phosphorylation Represses Protein Kinase R Activity.
Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J
2017-03-10
The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.
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Emerging roles and regulation of MiT/TFE transcriptional factors.
Yang, Min; Liu, En; Tang, Li; Lei, Yuanyuan; Sun, Xuemei; Hu, Jiaxi; Dong, Hui; Yang, Shi-Ming; Gao, Mingfa; Tang, Bo
2018-06-15
The MiT/TFE transcription factors play a pivotal role in the regulation of autophagy and lysosomal biogenesis. The subcellular localization and activity of MiT/TFE proteins are primarily regulated through phosphorylation. And the phosphorylated protein is retained in the cytoplasm and subsequently translocates to the nucleus upon dephosphorylation, where it stimulates the expression of hundreds of genes, leading to lysosomal biogenesis and autophagy induction. The transcription factor-mediated lysosome-to-nucleus signaling can be directly controlled by several signaling molecules involved in the mTORC1, PKC, and AKT pathways. MiT/TFE family members have attracted much attention owing to their intracellular clearance of pathogenic factors in numerous diseases. Recently, multiple studies have also revealed the MiT/TFE proteins as master regulators of cellular metabolic reprogramming, converging on autophagic and lysosomal function and playing a critical role in cancer, suggesting that novel therapeutic strategies could be based on the modulation of MiT/TFE family member activity. Here, we present an overview of the latest research on MiT/TFE transcriptional factors and their potential mechanisms in cancer.
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A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice
Illendula, Anuradha; Pulikkan, John A.; Zong, Hongliang; Grembecka, Jolanta; Xue, Liting; Sen, Siddhartha; Zhou, Yunpeng; Boulton, Adam; Kuntimaddi, Aravinda; Gao, Yan; Rajewski, Roger A.; Guzman, Monica L.; Castilla, Lucio H.; Bushweller, John H.
2015-01-01
Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers. PMID:25678665
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Lee, Sang Hyun; McCormick, Frank
2006-01-01
p97 (also referred to as DAP5, NAT1 or eIF4G2) has been proposed to act as a repressor of protein synthesis. However, we found that p97 is abundantly expressed in proliferating cells and p97 is recruited to ribosomes following growth factor stimulation. We also report that p97 binds eIF2β through its C-terminal domain and localizes to ribosome through its N-terminal MIF4G domain. When overexpressed, p97 increases reporter luciferase activity. In contrast, overexpression of the C-terminal two-thirds of eukaryotic initiation factor 4GI (eIF4GI), a region that shares significant homology with p97, or the N-terminal MIF4G domain of p97 markedly inhibits reporter activity, the rate of global translation and cell proliferation. Conversely, downregulation of p97 levels by RNA interference also decreases the rate of global translation and inhibits cell proliferation. This coincides with an increase in p27/Kip1 protein levels and a marked decrease in CDK2 kinase activity. Taken together, our results demonstrate that p97 is functionally different from the closely related C-terminal two-thirds of eIF4GI and it can positively promote protein synthesis and cell proliferation. PMID:16932749
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Klein, O; Kegler-Ebo, D; Su, J; Smith, S; DiMaio, D
1999-04-01
The bovine papillomavirus E5 gene encodes a 44-amino-acid, homodimeric transmembrane protein that is the smallest known transforming protein. The E5 protein transforms cultured fibroblasts by forming a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, leading to sustained receptor activation. Aspartic acid 33 in the extracellular juxtamembrane region of the E5 protein is important for cell transformation and interaction with the PDGF beta receptor. A. N. Meyer et al. (Proc. Natl. Acad. Sci USA 91:4634-4638, 1994) speculated that this residue interacted with lysine 499 on the receptor. We constructed E5 mutants containing all possible substitutions at position 33, as well as several double mutants containing substitutions at aspartic acid 33 and at glutamic acid 36, and we examined the ability of these mutants to transform C127 mouse fibroblasts and to bind to and induce activation of the PDGF beta receptor. There was an excellent correlation between the transformation activities of the various mutants and their ability to bind to and activate the PDGF beta receptor. Analysis of the mutants demonstrated that a juxtamembrane negative charge on the E5 protein was required for cell transformation and for productive interaction with the PDGF beta receptor and indicated that aspartic acid 33 was more important for these activities than was glutamic acid 36. These results are consistent with the existence of an essential juxtamembrane salt bridge between lysine 499 on the PDGF beta receptor and an acidic residue in the C terminus of the E5 protein and lend support to our proposed model for the complex between the E5 dimer and the PDGF beta receptor.
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Ma, Quan; Ying, Ming; Sui, Xiaojing; Zhang, Huimin; Huang, Haiyan; Yang, Linqing; Huang, Xinfeng; Zhuang, Zhixiong; Liu, Jianjun; Yang, Xifei
2015-01-01
Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway.
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Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian
2011-01-01
The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299
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Evaluation of hyaluronic acid-protein conjugates for polymer masked-unmasked protein therapy.
Ferguson, Elaine L; Alshame, Alshame M J; Thomas, David W
2010-12-15
Bioresponsive polymers may effectively be utilized to enhance the circulation time and stability of biologically active proteins and peptides, while reducing their immunogenicity and toxicity. Recently, dextrin-epidermal growth factor (EGF) conjugates, which make use of the Polymer-masked UnMasked Protein Therapy (PUMPT) concept, have been developed and shown potential as modulators of impaired wound healing. This study investigated the potential of PUMPT using hyaluronic acid (HA) conjugates to mask activity and enhance protein stability, while allowing restoration of biological activity following triggered degradation. HA fragments (Mw ∼90,000g/mol), obtained by acid hydrolysis of Rooster comb HA, were conjugated to trypsin as a model enzyme or to EGF as a model growth factor. Conjugates contained 2.45 and 0.98% (w/w) trypsin or EGF, respectively, and contained <5% free protein. HA conjugation did not significantly alter trypsin's activity. However, incubation of the conjugate with physiological concentrations of HAase increased its activity to ∼145% (p<0.001) that of the free enzyme. In contrast, when HA-EGF conjugates were tested in vitro, no effect on cell proliferation was seen, even in the presence of HAase. HA conjugates did not display typical masking/unmasking behavior, HA-trypsin conjugates exhibited ∼52% greater stability in the presence of elastase, compared to free trypsin, demonstrating the potential of HA conjugates for further development as modulators of tissue repair. Copyright © 2010 Elsevier B.V. All rights reserved.
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New Epigenetic Therapeutic Intervention for Metastatic Breast Cancer
2016-04-01
also discovered that constitutively activated NF -kB in TNBC sustains prolonged activation of pro-inflammatory cytokines, enabling rapid spread...metastasis) of TNBC tumors. Notably, the functions of both transcription factors Twist and NF -kB in gene activation require lysine acetylation, which signs...proteins including Twist, NF -kB and STAT3. b. Define the molecular basis of the BET BrDs’ selective interactions with effector proteins through structure
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Inoue, Kazuaki; Tsukiyama-Kohara, Kyoko; Matsuda, Chiho; Yoneyama, Mitsutoshi; Fujita, Takashi; Kuge, Shusuke; Yoshiba, Makoto; Kohara, Michinori
2012-11-30
Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-β induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-β mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-β mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Copyright © 2012 Elsevier Inc. All rights reserved.
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Assignment of the zinc ligands in RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor.
Zdanowski, Konrad; Doughty, Phillip; Jakimowicz, Piotr; O'Hara, Liisa; Buttner, Mark J; Paget, Mark S B; Kleanthous, Colin
2006-07-11
ZAS proteins are widespread bacterial zinc-containing anti-sigma factors that regulate the activity of sigma factors in response to diverse cues. One of the best characterized ZAS proteins is RsrA from Streptomyces coelicolor, which responds to disulfide stress. Zn-RsrA binds and represses the transcriptional activity of sigmaR in the reducing environment of the cytoplasm but undergoes reversible, intramolecular disulfide bond formation during oxidative stress. This expels the single metal ion and causes dramatic structural changes in RsrA that result in its dissociation from sigmaR, leaving the sigma factor free to activate the transcription of antioxidant genes. We showed recently that Zn2+ serves a critical role in modulating the redox activity of RsrA thiols but uncertainty remains as to how the metal ion is coordinated in RsrA and related ZAS proteins. Using a combination of random and site-specific mutagenesis with zinc K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy, we have assigned unambiguously the metal ligands in RsrA, thereby distinguishing between the different ligation models that have been proposed. The data show that the zinc site in RsrA is comprised of Cys11, His37, Cys41, and Cys44. Three of these residues are part of a conserved ZAS-specific sequence motif (H37xxxC41xxC44), with the fourth ligand, Cys11, found in a subset of ZAS proteins. Cys11 and Cys44 form the trigger disulfide in RsrA, explaining why the metal ion is expelled during oxidation. We discuss these data in the context of redox sensing by RsrA and the sensory mechanisms of other ZAS proteins.
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Silva, Ana F; Carvalho, Gilda; Soares, Renata; Coelho, Ana V; Barreto Crespo, M Teresa
2012-08-01
Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4-12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25-32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples.
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Ling, L; Kung, H J
1995-01-01
Nyk/Mer is a recently identified receptor tyrosine kinase with neural cell adhesion molecule-like structure (two immunoglobulin G-like domains and two fibronectin III-like domains) in its extracellular region and belongs to the Ufo/Axl family of receptors. The ligand for Nyk/Mer is presently unknown, as are the signal transduction pathways mediated by this receptor. We constructed and expressed a chimeric receptor (Fms-Nyk) composed of the extracellular domain of the human colony-stimulating factor 1 receptor (Fms) and the transmembrane and cytoplasmic domains of human Nyk/Mer in NIH 3T3 fibroblasts in order to investigate the mitogenic signaling and biochemical properties of Nyk/Mer. Colony-stimulating factor 1 stimulation of the Fms-Nyk chimeric receptor in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a proliferative response in the absence of other growth factors. We show that phospholipase C gamma, phosphatidylinositol 3-kinase/p70 S6 kinase, Shc, Grb2, Raf-1, and mitogen-activated protein kinase are downstream components of the Nyk/Mer signal transduction pathways. In addition, Nyk/Mer weakly activates p90rsk, while stress-activated protein kinase, Ras GTPase-activating protein (GAP), and GAP-associated p62 and p190 proteins are not activated or tyrosine phosphorylated by Nyk/Mer. An analysis comparing the Nyk/Mer signal cascade with that of the epidermal growth factor receptor indicates substrate preferences by these two receptors. Our results provide a detailed description of the Nyk/Mer signaling pathways. Given the structural similarity between the Ufo/Axl family receptors, some of the information may also be applied to other members of this receptor tyrosine kinase family. PMID:8524223
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Polyanion-Induced Self Association of Complement Factor H1
Pangburn, Michael K.; Rawal, Nenoo; Cortes, Claudio; Alam, M. Nurul; Ferreira, Viviana P.; Atkinson, Mark A. L.
2008-01-01
Factor H is the primary soluble regulator of activation of the alternative pathway of complement. It prevents activation of complement on host cells and tissues upon association with C3b and surface polyanions such as sialic acids, heparin and other glycosaminoglycans. Here we show that interaction with polyanions causes self-association forming tetramers of the 155,000 Da glycosylated protein. Monomeric human factor H is an extended flexible protein that exhibits an apparent size of 330,000 Da, relative to globular standards, during gel filtration chromatography in the absence of polyanions. In the presence of dextran sulfate (5,000 Da) or heparin an intermediate species of apparent m.w. 700,000 and a limit species of m.w. 1,400,000 were observed by gel filtration. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a Mr of 607,000, corresponding to a tetramer, in the presence of less than a 2-fold molar excess of dextran sulfate. Increasing concentrations of dextran sulfate increased binding of factor H to zymosan-C3b 4.5-fold. This was accompanied by an increase in both the decay accelerating and cofactor activity of factor H on these cells. An expressed fragment encompassing the C-terminal polyanion binding site (complement control protein domains 18–20) also exhibited polyanion-induced self association, suggesting that the C-terminal ends of factor H mediate self-association. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H, and the resulting regulation of complement activation, may involve formation of dimers and tetramers of factor H. PMID:19124749
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Abstract
Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus, birds and mammals. Dimers of Rel proteins form the transcription factor nuclear factor
AP1 Keeps Chromatin Poised for Action | Center for Cancer Research
The human genome harbors gene-encoding DNA, the blueprint for building proteins that regulate cellular function. Embedded across the genome, in non-coding regions, are DNA elements to which regulatory factors bind. The interaction of regulatory factors with DNA at these sites modifies gene expression to modulate cell activity. In cells, DNA exists in a complex with proteins
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Tokunaga, Masayuki; Baron, Byron; Kitagawa, Takao; Tokuda, Kazuhiro; Kuramitsu, Yasuhiro
2015-11-01
Active hexose-correlated compound (AHCC) is an extract of a basidiomycete mushroom that enhances the therapeutic effects and reduces the side-effects of chemotherapy. Our previous studies demonstrated that heat-shock protein 27 (HSP27) was involved in gemcitabine-resistance of pancreatic cancer cells and it was down-regulated by AHCC-treatment. However, how AHCC down-regulated HSP27 is unknown. In the present study, we focused on two transcription factors reported to induce HSP27, heat shock factor 1 (HSF1) and high-mobility group box 1 (HMGB1) and investigated the effect of AHCC on their expression. KLM1-R cells were treated with AHCC and the protein expression of HSF1 and HMGB1 were analyzed by western blotting. The protein expression of HSF1 in KLM1-R was down-regulated by AHCC treatment. On the other hand, the protein expression of HMGB1 was not reduced in KLM1-R cells after AHCC treatment. The possibility that AHCC down-regulated HSP27 through down-regulation of the HSF1, was herein shown. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.
Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela
2017-09-06
In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ogura, Hirotsugu; Tsukumo, Yoshinori; Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501
2008-04-01
The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in amore » dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-{alpha}-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8.« less
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Schenning, Martijn; Goedhart, Joachim; Gadella, Theodorus W J; Avram, Diana; Wirtz, Karel W A; Snoek, Gerry T
2008-10-01
The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein alpha (PI-TPalpha; SPIalpha cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555-1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIalpha>wtNIH3T3>SPIbeta cells (i.e. wild type cells overexpressing PI-TPbeta). Upon incubation of SPIbeta cells with the PI-TPalpha-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-kappaB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIalpha cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIbeta cells as visualized by confocal laser scanning microscopy.
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Van Snick, J; Houssiau, F; Proost, P; Van Damme, J; Renauld, J C
1996-09-15
We have previously reported that cytokines such as IL-9, IL-4, and IL-6 protect murine thymic lymphoma cell lines against dexamethasone-induced apoptosis. A similar activity, which could not be ascribed to any of these factors, was found in a number of human T cell supernatants that enabled mouse BW5147 thymic lymphoma not only to escape apoptosis but also to maintain proliferation. The protein responsible for this activity was purified to homogeneity from the culture medium of activated leukemic T cells and was found to be identical with the I-309 chemokine. Half-maximal anti-apoptotic activity was obtained with approximately 1 ng/ml, a concentration considerably lower than that required for the monocyte chemotactic activity of this molecule, as measured on THP-1 cells. The purified I-309 also improved the survival of two other mouse thymic lymphoma cell lines. This activity was as potent as that of IL-9, which was the strongest anti-apoptotic factor found to date for these cells. Similar results were obtained for BW5147 cells with recombinant I-309 and with T cell activation gene-3, the murine homologue of I-309, but not with other members of the chemokine family, including IL-8, neutrophil-activating peptide-2, granulocyte chemotactic protein-2, macrophage inflammatory protein-1a, RANTES (regulated upon activation, normal T cell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), and MCP-2. MCP-3, however, showed a minor, but significant effect in this model. Unlike that of IL-9, the activity of I-309 was completely inhibited in the presence of pertussis toxin, indicating the involvement of a G protein in this process.
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Papaioannou, Anastasios; Kuyucak, Serdar; Kuncic, Zdenka
2016-01-01
The insulin-family proteins bind to their own receptors, but insulin-like growth factor II (IGF-II) can also bind to the A isoform of the insulin receptor (IR-A), activating unique and alternative signaling pathways from those of insulin. Although extensive studies of insulin have revealed that its activation is associated with the opening of the B chain-C terminal (BC-CT), the activation mechanism of the insulin-like growth factors (IGFs) still remains unknown. Here, we present the first comprehensive study of the insulin-family proteins comparing their activation process and mechanism using molecular dynamics simulations to reveal new insights into their specificity to the insulin receptor. We have found that all the proteins appear to exhibit similar stochastic dynamics in their conformational change to an active state. For the IGFs, our simulations show that activation involves two opening locations: the opening of the BC-CT section away from the core, similar to insulin; and the additional opening of the BC-CT section away from the C domain. Furthermore, we have found that these two openings occur simultaneously in IGF-I, but not in IGF-II, where they can occur independently. This suggests that the BC-CT section and the C domain behave as a unified domain in IGF-I, but as two independent domains in IGF-II during the activation process, implying that the IGFs undergo different activation mechanisms for receptor binding. The probabilities of the active and inactive states of the proteins suggest that IGF-II is hyperactive compared to IGF-I. The hinge residue and the hydrophobic interactions in the core are found to play a critical role in the stability and activity of IGFs. Overall, our simulations have elucidated the crucial differences and similarities in the activation mechanisms of the insulin-family proteins, providing new insights into the molecular mechanisms responsible for the observed differences between IGF-I and IGF-II in receptor binding.
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Mitogen-activated protein kinase inhibition reduces mucin 2 production and mucinous tumor growth.
Dilly, Ashok K; Song, Xinxin; Zeh, Herbert J; Guo, Zong S; Lee, Yong J; Bartlett, David L; Choudry, Haroon A
2015-10-01
Excessive accumulation of mucin 2 (MUC2) protein (a gel-forming secreted mucin) within the peritoneal cavity is the major cause of morbidity and mortality in pseudomyxoma peritonei (PMP), a unique mucinous malignancy of the appendix. Mitogen-activated protein kinase (MAPK) signaling pathway is upregulated in PMP and has been shown to modulate MUC2 promoter activity. We hypothesized that targeted inhibition of the MAPK pathway would be a novel, effective, and safe therapeutic strategy to reduce MUC2 production and mucinous tumor growth. We tested RDEA119, a specific MEK1/2 (MAPK extracellular signal-regulated kinase [ERK] kinase) inhibitor, in MUC2-secreting LS174T cells, human PMP explant tissue, and in a unique intraperitoneal murine xenograft model of PMP. RDEA119 reduced ERK1/2 phosphorylation and inhibited MUC2 messenger RNA and protein expression in vitro. In the xenograft model, chronic oral therapy with RDEA119 inhibited mucinous tumor growth in an MAPK pathway-dependent manner and this translated into a significant improvement in survival. RDEA119 downregulated phosphorylated ERK1/2 and nuclear factor κB p65 protein signaling and reduced activating protein 1 (AP1) transcription factor binding to the MUC2 promoter in LS174T cells. This study provides a preclinical rationale for the use of MEK inhibitors to treat patients with PMP. Copyright © 2015 Elsevier Inc. All rights reserved.
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Fisher, Susan H; Wray, Lewis V
2008-01-22
The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS-GlnR-DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein-protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR-DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein-protein interaction that stabilizes GlnR-DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhang, Yingyi; Zhao, Yu; Li, Leilei
2013-05-03
Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB)more » plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.« less
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Sueoka, E; Nishiwaki, S; Okabe, S; Iida, N; Suganuma, M; Yano, I; Aoki, K; Fujiki, H
1995-08-01
Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales, such as Mycobacterium, Nocardia and Rhodococcus. They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage-derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis, trehalose 6-monomycolate (mTMM) and trehalose 6,6'-dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca2+, and mTMM activated PKC alpha more strongly than PKC beta or gamma under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca2+ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor-alpha (TNF-alpha) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF-alpha is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed.
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Ohlmann, Philippe; Hechler, Béatrice; Chafey, Philippe; Ravanat, Catherine; Isola, Hervé; Wiesel, Marie-Louise; Cazenave, Jean-Pierre; Gachet, Christian
2016-09-01
The INTERCEPT Blood System (IBS) using amotosalen-HCl and ultraviolet (UV)A inactivates a large spectrum of microbial pathogens and white blood cells in therapeutic plasma. Our aim was to evaluate to what extent IBS modifies the capacity of plasma to generate thrombin and induces qualitative or quantitative modifications of plasma proteins. Plasma units from four donors were collected by apheresis. Samples were taken before (control [CTRL]) and after IBS treatment and stored at -80°C until use. The activities of plasma coagulation factors and inhibitors and the thrombin generation potential were determined using assays measuring clotting times and the calibrated automated thrombogram (CAT), respectively. The proteomic profile of plasma proteins was examined using a two-dimensional differential in-gel electrophoresis (2D-DIGE) method. Nearly all of the procoagulant and antithrombotic factors tested retained at least 78% of their initial pre-IBS activity. Only FVII and FVIII displayed a lower level of conservation (67%), which nevertheless remained within the reference range for conventional plasma coagulation factors. The thrombin generation profile of plasma was conserved after IBS treatment. Among the 1331 protein spots revealed by 2D-DIGE analysis, only four were differentially expressed in IBS plasma compared to CTRL plasma and two were identified by mass spectrometric analysis as transthyretin and apolipoprotein A1. The IBS technique for plasma moderately decreases the activities of plasma coagulation factors and antithrombotic proteins, with no impact on the thrombin generation potential of plasma and very limited modifications of the proteomic profile. © 2016 AABB.
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Tanner, Julie-Anne; Prasad, Bhagwat; Claw, Katrina G; Stapleton, Patricia; Chaudhry, Amarjit; Schuetz, Erin G; Thummel, Kenneth E; Tyndale, Rachel F
2017-01-01
Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
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Borges, Chad R; Rehder, Douglas S
2016-09-15
Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.
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Zaret, K S; Watts, J; Xu, J; Wandzioch, E; Smale, S T; Sekiya, T
2008-01-01
The endoderm is a multipotent progenitor cell population in the embryo that gives rise to the liver, pancreas, and other cell types and provides paradigms for understanding cell-type specification. Studies of isolated embryo tissue cells and genetic approaches in vivo have defined fibroblast growth factor/mitogen-activated protein kinase (FGF/MAPK) and bone morphogenetic protein (BMP) signaling pathways that induce liver and pancreatic fates in the endoderm. In undifferentiated endoderm cells, the FoxA and GATA transcription factors are among the first to engage silent genes, helping to endow competence for cell-type specification. FoxA proteins can bind their target sites in highly compacted chromatin and open up the local region for other factors to bind; hence, they have been termed "pioneer factors." We recently found that FoxA proteins remain bound to chromatin in mitosis, as an epigenetic mark. In embryonic stem cells, which lack FoxA, FoxA target sites can be occupied by FoxD3, which in turn helps to maintain a local demethylation of chromatin. By these means, a cascade of Fox factors helps to endow progenitor cells with the competence to activate genes in response to tissue-inductive signals. Understanding such epigenetic mechanisms for transcriptional competence coupled with knowledge of the relevant signals for cell-type specification should greatly facilitate efforts to predictably differentiate stem cells to liver and pancreatic fates.
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Liu, Xinhua; Pan, Lilong; Wang, Xianli; Gong, Qihai; Zhu, Yi Zhun
2012-05-01
Leonurine, a bioactive alkaloid compound in Herba leonuri, has various pharmacological activities, including antioxidant and anti-apoptotic capacities. This study was conducted to test the hypothesis that leonurine was able to attenuate tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) activation and the underlying molecular mechanisms. Mitogen-activated protein kinases (MAPK) activation, nuclear factor-κB (NF-κB) activation, and inflammatory mediators expression were detected by Western blot or enzyme-liked immunosorbent assay, intracellular reactive oxygen species (ROS) and NF-κB p65 translocation were measured by immunofluorescence, endothelial cell-monocyte interaction was detected by microscope. Leonurine inhibited U937 cells adhesion to TNF-α-activated HUVEC in a concentration dependent manner. Treatment with leonurine blocked TNF-α-induced mRNA and protein expression of adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), cyclooxygenase-2, and monocyte chemoattractant protein-1 in endothelial cells. In addition, leonurine attenuated TNF-α-induced intracellular ROS production in HUVEC. Furthermore, leonurine also suppressed the TNF-α-activated p38 phosphorylation and IκBα degradation. Subsequently, reduced NF-κB p65 phosphorylation, nuclear translocation, and DNA-binding activity were also observed. Our results demonstrated for the first time that the anti-inflammatory properties of leonurine in endothelial cells, at least in part, through suppression of NF-κB activation, which may have a potential therapeutic use for inflammatory vascular diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
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Exploring the roles of basal transcription factor 3 in eukaryotic growth and development.
Jamil, Muhammad; Wang, Wenyi; Xu, Mengyun; Tu, Jumin
2015-01-01
Basal transcription factor 3 (BTF3) has been reported to play a significant part in the transcriptional regulation linking with eukaryotes growth and development. Alteration in the BTF3 gene expression patterns or variation in their activities adds to the explanation of different signaling pathways and regulatory networks. Moreover, BTF3s often respond to numerous stresses, and subsequently they are involved in regulation of various mechanisms. BTF3 proteins also function through protein-protein contact, which can assist us to identify the multifaceted processes of signaling and transcriptional regulation controlled by BTF3 proteins. In this review, we discuss current advances made in starting to explore the roles of BTF3 transcription factors in eukaryotes especially in plant growth and development.