scholarly journals Role of Citrate and Phosphate Anions in the Mechanism of Iron(III) Sequestration by Ferric Binding Protein: Kinetic Studies of the Formation of the Holoprotein of Wild-Type FbpA and Its Engineered Mutants

Biochemistry ◽  
2010 ◽  
Vol 49 (29) ◽  
pp. 6021-6032 ◽  
Author(s):  
Katherine D. Weaver ◽  
Mario Gabričević ◽  
Damon S. Anderson ◽  
Pratima Adhikari ◽  
Timothy A. Mietzner ◽  
...  
Keyword(s):  
Binding Protein ◽  
Kinetic Studies ◽  
Wild Type ◽  
Ferric Binding Protein ◽  
Phosphate Anions

scholarly journals C4b binding protein negatively regulates TLR1/2 response

2016 ◽  
Vol 23 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Naoko Morita ◽  
Ikuko Yamai ◽  
Koichiro Takahashi ◽  
Yutaka Kusumoto ◽  
Takuma Shibata ◽  
...  
Keyword(s):  
Inflammatory Cytokine ◽  
Cytokine Production ◽  
Binding Protein ◽  
Negative Regulation ◽  
Negative Regulator ◽  
Wild Type ◽  
Inflammatory Cytokine Production ◽  
Inflammatory Signals ◽  
Deficient Mice

TLR2 associates with TLR1 and recognizes microbial lipoproteins. Pam3CSK4, a triacylated lipoprotein, is anchored to the extracellular domain of TLR1 and TLR2 and induces pro-inflammatory signals. Here we show that C4b binding protein (C4BP), which is a complement pathway inhibitor, is a TLR2-associated molecule. Immunoprecipitation assay using anti-TLR2 mAb shows that C4BP binds to TLR2. In C4BP-deficient mice, Pam3CSK4-induced IL-6 levels were increased compared with wild type mice. In C4BP-expressing cells, Pam3CSK4-induced IL-8 production was reduced depending on the C4BP expression levels. These results reveal the important role of C4BP in negative regulation of TLR1/2-dependent pro-inflammatory cytokine production. Furthermore, using a fluorescent conjugated Pam3CSK4, we show that C4BP blocks the binding of Pam3CSK4 to TLR1/2. Finally, we show that exogenous C4BP also inhibits Pam3CSK4-induced signaling leading to IL-8 production. Our results indicate C4BP binding to TLR2 and consequent neutralization of its activity otherwise inducing pro-inflammatory cytokine production. C4BP is a negative regulator of TLR1/2 activity.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Essential Role of Insulin Receptor Substrate 1 (IRS-1) and IRS-2 in Adipocyte Differentiation

2001 ◽  
Vol 21 (7) ◽  
pp. 2521-2532 ◽  
Author(s):  
Hiroshi Miki ◽  
Toshimasa Yamauchi ◽  
Ryo Suzuki ◽  
Kajuro Komeda ◽  
Atsuko Tsuchida ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Adipocyte Differentiation ◽  
Kinase Inhibitor ◽  
Binding Protein ◽  
Insulin Receptor Substrate ◽  
Wild Type ◽  
Double Knockout ◽  
Insulin Receptor Substrate 1 ◽  
Fatty Acid Binding

ABSTRACT To investigate the role of insulin receptor substrate 1 (IRS-1) and IRS-2, the two ubiquitously expressed IRS proteins, in adipocyte differentiation, we established embryonic fibroblast cells with four different genotypes, i.e., wild-type, IRS-1 deficient (IRS-1−/−), IRS-2 deficient (IRS-2−/−), and IRS-1 IRS-2 double deficient (IRS-1−/−IRS-2−/−), from mouse embryos of the corresponding genotypes. The abilities of IRS-1−/− cells and IRS-2−/− cells to differentiate into adipocytes are approximately 60 and 15%, respectively, lower than that of wild-type cells, at day 8 after induction and, surprisingly, IRS-1−/− IRS-2−/− cells have no ability to differentiate into adipocytes. The expression of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) is severely decreased in IRS-1−/−IRS-2−/− cells at both the mRNA and the protein level, and the mRNAs of lipoprotein lipase and adipocyte fatty acid binding protein are severely decreased in IRS-1−/−IRS-2−/− cells. Phosphatidylinositol 3-kinase (PI 3-kinase) activity that increases during adipocyte differentiation is almost completely abolished in IRS-1−/−IRS-2−/− cells. Treatment of wild-type cells with a PI 3-kinase inhibitor, LY294002, markedly decreases the expression of C/EBPα and PPARγ, a result which is associated with a complete block of adipocyte differentiation. Moreover, histologic analysis of IRS-1−/− IRS-2−/− double-knockout mice 8 h after birth reveals severe reduction in white adipose tissue mass. Our results suggest that IRS-1 and IRS-2 play a crucial role in the upregulation of the C/EBPα and PPARγ expression and adipocyte differentiation.

scholarly journals The Unique Cyanobacterial Protein OpcA Is an Allosteric Effector of Glucose-6-phosphate Dehydrogenase inNostoc punctiformeATCC 29133

2001 ◽  
Vol 276 (15) ◽  
pp. 11477-11486 ◽  
Author(s):  
Kari D. Hagen ◽  
John C. Meeks
Keyword(s):  
Kinetic Studies ◽  
G6pd Activity ◽  
Wild Type ◽  
Nostoc Punctiforme ◽  
Aggregation State ◽  
Allosteric Effector ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Tetrameric Form ◽  
Allosteric Activator

Glucose-6-phosphate dehydrogenase (G6PD), encoded byzwf, is essential for nitrogen fixation and dark heterotrophic growth of the cyanobacteriumNostoc punctiformeATCC 29133. InN. punctiforme,zwfis part of a four-gene operon transcribed in the orderfbp-tal-zwf-opcA. Genetic analyses indicated thatopcAis required for G6PD activity. To define the role ofopcA, the synthesis, aggregation state, and activity of G6PD inN. punctiformestrains expressing different amounts of G6PD and/or OpcA were examined. A single tetrameric form of G6PD was consistently observed for all strains, as well as for recombinantN. punctiformeHis-G6PD purified fromEscherichia coli, regardless of the quantity of OpcA present. However, His-G6PD and the G6PD of strain UCD 351, which lacks OpcA, had low affinities for glucose 6-phosphate (G6P) substrate (Km(app) = 65 and 85 mm, respectively) relative to wild-typeN. punctiformeG6PD (Km(app) = 0.5 mm). Near wild-type affinities for G6P were observed for these enzymes when saturating amounts of His-OpcA- or OpcA-containing extract were added. Kinetic studies were consistent with OpcA acting as an allosteric activator of G6PD. A role in redox modulation of G6PD activity was also indicated, because thioredoxin-mediated inactivation and reactivation of His-G6PD occurred only when His-OpcA was present.

scholarly journals Transplacental delivery of retinoid: the role of retinol-binding protein and lipoprotein retinyl ester

2004 ◽  
Vol 286 (5) ◽  
pp. E844-E851 ◽  
Author(s):  
Loredana Quadro ◽  
Leora Hamberger ◽  
Max E. Gottesman ◽  
Vittorio Colantuoni ◽  
Rajasekhar Ramakrishnan ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Congenital Malformations ◽  
Binding Protein ◽  
Retinol Binding Protein ◽  
Wild Type ◽  
Maternal Circulation ◽  
Retinyl Ester ◽  
Retinyl Esters ◽  
Normal Embryonic Development

Retinoids are required for normal embryonic development. Both embryonic retinoid deficiency and excess result in congenital malformations. There is little understanding of the physiology underlying retinoid transfer from the maternal circulation to the embryo. We now report studies that explore this process using retinol-binding protein-deficient (RBP−/−) mice and mice that express human RBP on the RBP−/−background. Our studies establish that dietary retinoid, bound to lipoproteins, can serve as an important source for meeting tissue retinoid requirements during embryogenesis. Indeed, retinyl ester concentrations in the circulations of pregnant RBP−/−mice are significantly elevated over those observed in wild-type mice, suggesting that lipoprotein retinyl esters may compensate for the absence of retinol-RBP during pregnancy. We also demonstrate, contrary to earlier proposals, that maternal RBP does not cross the placenta and cannot enter the fetal circulation. Overall, our data indicate that both retinol-RBP and retinyl esters bound to lipoproteins are able to provide sufficient retinoid to the embryo to allow for normal embryonic development.

scholarly journals Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration

Blood ◽  
2012 ◽  
Vol 119 (3) ◽  
pp. 838-846 ◽  
Author(s):  
John C. Kostyak ◽  
Meghna U. Naik ◽  
Ulhas P. Naik
Keyword(s):  
Mouse Model ◽  
Binding Protein ◽  
Dual Role ◽  
Wild Type ◽  
Platelet Recovery ◽  
Polyploid Cells ◽  
And Migration ◽  
Platelet Depletion

Abstract Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1−/− mouse model. Cib1−/− mice have more platelets and BM megakaryocytes than wild-type (WT) controls (P < .05). Furthermore, subsequent analysis of megakaryocyte-CFU production revealed an increase with Cib1 deletion compared with WT (P < .05). In addition, BM from Cib1−/− mice, cultured with thrombopoietin (TPO) for 24 hours, produced more highly polyploid megakaryocytes than WT BM (P < .05). Subsequent analysis of TPO signaling revealed enhanced Akt and ERK1/2 phosphorylation, whereas FAKY925 phosphorylation was reduced in Cib1−/− megakaryocytes treated with TPO. Conversely, platelet recovery in Cib1−/− mice after platelet depletion was attenuated compared with WT (P < .05). This could be the result of impaired adhesion and migration, as adhesion to fibrinogen and fibronectin and migration toward an SDF-1α gradient were reduced in Cib1−/− megakaryocytes compared with WT (P < .05). In addition, Cib1−/− megakaryocytes formed fewer proplatelets compared with WT (P < .05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in megakaryopoiesis, initially by negatively regulating TPO signaling and later by augmenting proplatelet production.

scholarly journals Synergistic Activation of the Prolactin Promoter by Vitamin D Receptor and GHF-1: Role of the Coactivators, CREB-Binding Protein and Steroid Hormone Receptor Coactivator-1 (SRC-1)

1999 ◽  
Vol 13 (7) ◽  
pp. 1141-1154 ◽  
Author(s):  
Ana I. Castillo ◽  
Ana M. Jimenez-Lara ◽  
Rosa M. Tolon ◽  
Ana Aranda
Keyword(s):  
Vitamin D ◽  
Vitamin D Receptor ◽  
Hormone Receptor ◽  
Binding Protein ◽  
Steroid Hormone ◽  
Steroid Hormone Receptor ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Prl Gene

Abstract PRL gene expression is dependent on the presence of the pituitary-specific transcription factor GHF-1/Pit-1, which is transcribed in a highly restricted manner in cells of the anterior pituitary. In pituitary GH3 cells, vitamin D increases the levels of PRL transcripts and stimulates the PRL promoter. We have analyzed the role of GHF-1 and of the vitamin D receptor (VDR) to confer vitamin D responsiveness to the PRL promoter. For this purpose we have used nonpituitary HeLa cells, which do not express GHF-1. We found that VDR activates the PRL promoter both in a ligand-dependent and -independent manner through a sequence located between positions− 45/−27 in the proximal 5′-flanking region. This sequence also confers VDR and vitamin D responsiveness to a heterologous promoter. In the context of the PRL gene, VDR requires the presence of GHF-1 to activate the promoter. Truncation of the last 12 C-terminal amino acids of VDR, which contain the ligand-dependent activation function (AF2), abolishes regulation by vitamin D, suggesting that binding of coactivators to this region mediates ligand-dependent stimulation of the PRL promoter by the receptor. Indeed, expression of the coactivators, steroid hormone receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP), significantly enhances the stimulatory effect of vitamin D mediated by the wild-type VDR but not by the AF2 mutant receptor. Furthermore, CBP also increases the activation of the PRL promoter by GHF-1 and the ligand-independent activation by both wild-type and mutant VDR.

Kinetics and Mechanism of Iron(III) Complexation by Ferric Binding Protein:  The Role of Phosphate†

Biochemistry ◽  
2004 ◽  
Vol 43 (19) ◽  
pp. 5811-5819 ◽  
Author(s):  
Mario Gabričević ◽  
Damon S. Anderson ◽  
Timothy A. Mietzner ◽  
Alvin L. Crumbliss
Keyword(s):  
Binding Protein ◽  
Kinetics And Mechanism ◽  
Ferric Binding Protein

scholarly journals The plastid-nucleus located DNA/RNA binding protein WHIRLY1 regulates microRNA-levels during stress

2017 ◽  
Author(s):  
Aleksandra Swida-Barteczka ◽  
Anja Krieger-Liszkay ◽  
Wolfgang Bilger ◽  
Ulrike Voigt ◽  
Götz Hensel ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Nuclear Gene ◽  
Retrograde Signaling ◽  
Wild Type ◽  
Transcriptional Responses ◽  
Stress Dependent

In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression (Grabowski et al., 2008; Foyer et al., 2014). In this study the putative involvement of WHIRLY1 in stress dependent retrograde signaling was investigated by comparison of barley (Hordeum vulgare L., cv. Golden Promise) wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1. In contrast to the wild type, the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress (Kruszka et al., 2012, Sunkar et al., 2012). The results support a central role of WHIRLY1 in retrograde signaling and underpin a so far underestimated role of microRNAs in this process.

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