scholarly journals Isolation and Properties of the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL

1999 ◽  
Vol 181 (15) ◽  
pp. 4461-4468 ◽  
Author(s):  
Tracy Money ◽  
Tamera Jones ◽  
Ray Dixon ◽  
Sara Austin
Keyword(s):  
Complex Formation ◽  
Azotobacter Vinelandii ◽  
Binding Protein ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Cell Extracts ◽  
Enhancer Binding Protein ◽  
Adenosine Nucleotides

ABSTRACT In Azotobacter vinelandii, activation ofnif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.

scholarly journals Protein-Protein Interactions in the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL fromAzotobacter vinelandii

2001 ◽  
Vol 183 (4) ◽  
pp. 1359-1368 ◽  
Author(s):  
Tracy Money ◽  
Jason Barrett ◽  
Ray Dixon ◽  
Sara Austin
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Azotobacter Vinelandii ◽  
Protein Complexes ◽  
Binding Protein ◽  
Limited Proteolysis ◽  
Regulatory System ◽  
Terminal Domain ◽  
Enhancer Binding Protein

ABSTRACT The enhancer binding protein NIFA and the sensor protein NIFL fromAzotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography.

scholarly journals The N-Terminal Domain of p73 Interacts with the CH1 Domain of p300/CREB Binding Protein and Mediates Transcriptional Activation and Apoptosis

2000 ◽  
Vol 20 (4) ◽  
pp. 1299-1310 ◽  
Author(s):  
Xiaoya Zeng ◽  
Xiaorong Li ◽  
Ashley Miller ◽  
Zhimin Yuan ◽  
Wuchao Yuan ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Binding Protein ◽  
Small Cell Lung Carcinoma ◽  
Creb Binding Protein ◽  
Cell Lung Carcinoma ◽  
Cell Extracts ◽  
N Terminus ◽  
Mcf 7

ABSTRACT The newly identified p53 homolog p73 mimics the transcriptional function of p53. We have investigated the regulation of p73's transcriptional activity by p300/CREB binding protein (CBP). p73-p300 complexes were identified in HeLa cell extracts by cofractionation and coimmunoprecipitation assays. The p73-p300 interaction was confirmed in vitro by glutathione S-transferase–protein association assays and in vivo by coimmunoprecipitating the overexpressed p300 and p73 in human p53-free small-cell lung carcinoma H1299 or osteosarcoma Saos-2 cells. The N terminus but not the N-terminal truncation of p73 bound to the CH1 domain (amino acids [aa] 350 to 450) of p300/CBP. Accordingly, this p73 N-terminal deletion was unable to activate transcription or to induce apoptosis. Overexpression of either p300 or CBP stimulated transcription mediated by p73 but not its N-terminally deleted mutant in vivo. The N-terminal fragment from aa 19 to 597, but not the truncated fragment from aa 242 to 1700 of p300, reduced p73-mediated transcription markedly. p73-dependent transcription or apoptosis was partially impaired in either p300- or CBP-deficient human breast carcinoma MCF-7 or H1299 cells, suggesting that both coactivators mediate transcription by p73 in cells. These results demonstrate that the N terminus of p73 directly interacts with the N-terminal CH1 domain of p300/CBP to activate transcription.

scholarly journals Alpha 2-macroglobulin binds and inhibits activated protein C

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2283-2290 ◽  
Author(s):  
H Hoogendoorn ◽  
CH Toh ◽  
ME Nesheim ◽  
AR Giles
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Metal Ion ◽  
Protein C ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Molecular Weight Complex

In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine- Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

scholarly journals Characterization of the Klebsiella aerogenes Urease Accessory Protein UreD in Fusion with the Maltose Binding Protein

2010 ◽  
Vol 192 (9) ◽  
pp. 2294-2304 ◽  
Author(s):  
Eric L. Carter ◽  
Robert P. Hausinger
Keyword(s):  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Maltose Binding Protein ◽  
Accessory Protein ◽  
Klebsiella Aerogenes ◽  
E Coli ◽  
Cell Extracts ◽  
Recombinant Cells

ABSTRACT Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a ΔureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (>670 kDa) that bound approximately 2.5 Ni2+ ions (Kd of ∼50 μM, where Kd is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (∼4 Zn2+ ions per protomer; Kd of 5 μM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.

scholarly journals The CBP Bromodomain and Nucleosome Targeting Are Required for Zta-Directed Nucleosome Acetylation and Transcription Activation

2003 ◽  
Vol 23 (8) ◽  
pp. 2633-2644 ◽  
Author(s):  
Zhong Deng ◽  
Chi-Ju Chen ◽  
Michaela Chamberlin ◽  
Fang Lu ◽  
Gerd A. Blobel ◽  
...  
Keyword(s):  
Complex Formation ◽  
Histone Acetylation ◽  
Protein Sequencing ◽  
Creb Binding Protein ◽  
Barr Virus ◽  
Amino Terminal ◽  
Viral Promoters ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV)-encoded lytic activator Zta is a bZIP protein that can stimulate nucleosomal histone acetyltransferase (HAT) activity of the CREB binding protein (CBP) in vitro. We now show that deletion of the CBP bromo- and C/H3 domains eliminates stimulation of nucleosomal HAT activity in vitro and transcriptional coactivation by Zta in transfected cells. In contrast, acetylation of free histones was not affected by the addition of Zta or by deletions in the bromo or C/H3 domain of CBP. Zta stimulated acetylation of oligonucleosomes assembled on supercoiled DNA and dinucleosomes assembled on linear DNA, but Zta-stimulated acetylation was significantly reduced for mononucleosomes. Western blotting and amino-terminal protein sequencing indicated that all lysine residues in the H3 and H4 amino-terminal tails were acetylated by CBP and enhanced by the addition of Zta. Histone acetylation was also dependent upon the Zta basic DNA binding domain, which could not be substituted with the homologous basic region of c-Fos, indicating specificity in the bZIP domain nucleosome binding function. Finally, we show that Zta and CBP colocalize to viral immediate-early promoters in vivo and that overexpression of Zta leads to a robust increase in H3 and H4 acetylation at various regions of the EBV genome in vivo. Furthermore, deletion of the CBP bromodomain reduced stable CBP-Zta complex formation and histone acetylation at Zta-responsive viral promoters in vivo. These results suggest that activator- and bromodomain-dependent targeting to oligonucleosomal chromatin is required for stable promoter-bound complex formation and transcription activity.

scholarly journals Lissencephaly-1 is a context-dependent regulator of the human dynein complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Janina Baumbach ◽  
Andal Murthy ◽  
Mark A McClintock ◽  
Carly I Dix ◽  
Ruta Zalyte ◽  
...  
Keyword(s):  
Complex Formation ◽  
Binding Protein ◽  
Cytoplasmic Dynein ◽  
Cargo Transport ◽  
Dynein Motor ◽  
Context Dependent ◽  
Shed Light ◽  
Dynamic Microtubule

The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.

Lipin1-Mediated Repression of Adipogenesis by Rutin

2016 ◽  
Vol 44 (03) ◽  
pp. 565-578 ◽  
Author(s):  
Yo-Han Han ◽  
Ji-Ye Kee ◽  
Jinbong Park ◽  
Dae-Seung Kim ◽  
Soyoung Shin ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Binding Protein ◽  
Related Factors ◽  
Fatty Acid Binding ◽  
Upstream Regulator ◽  
Enhancer Binding Protein ◽  
Adipogenic Effect

Rutin, also called rutoside or quercetin-3-O-rutinoside and sophorin, is a glycoside between the flavonol quercetin and the disaccharide rutinose. Although many effects of rutin have been reported in vitro and in vivo, the anti-adipogenic effects of rutin have not been fully reported. The aim of this study was to confirm how rutin regulates adipocyte related factors. In this study, rutin decreased the expressions of adipogenesis-related genes, including peroxisome proliferators, activated receptor [Formula: see text] (PPAR[Formula: see text], CCAAT/enhancer-binding protein [Formula: see text] (C/EBP[Formula: see text], fatty acid synthase, adipocyte fatty acid-binding protein, and lipoprotein lipase in 3T3-L1 cells. Rutin also repressed the expression of lipin1, which is an upstream regulator that controls PPAR[Formula: see text] and C/EBP[Formula: see text]. In addition, when 3T3-L1 was transfected with lipin1 siRNA to block lipin1 function, rutin did not affect the expressions of PPAR[Formula: see text] and C/EBP[Formula: see text]. These results suggest that rutin has an anti-adipogenic effect that acts through the suppression of lipin1, as well as PPAR[Formula: see text] and C/EBP[Formula: see text].

scholarly journals Detection and characterization of a 3' untranslated region ribonucleoprotein complex associated with human alpha-globin mRNA stability.

1995 ◽  
Vol 15 (3) ◽  
pp. 1769-1777 ◽  
Author(s):  
X Wang ◽  
M Kiledjian ◽  
I M Weiss ◽  
S A Liebhaber
Keyword(s):  
Complex Formation ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Binding Protein ◽  
Erythroid Cell ◽  
Globin Mrna ◽  
Rnp Complex ◽  
Alpha Globin

The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human alpha-globin mRNA 3' untranslated region (alpha-3'UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the alpha-3'UTR with a variety of cytosolic extracts. This so-called alpha-complex is sequence specific and is not formed on the 3'UTR of either beta-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize alpha-globin mRNA in vivo result in a parallel disruption of the alpha-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of alpha-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the alpha-complex. Three predominant proteins are isolated by alpha-3'UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for alpha-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the alpha-3'UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of alpha-globin mRNA.

CDDO Increases Translation of CCAAT Enhancer Binding Protein alpha To Induce Granulocytic Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2458-2458 ◽  
Author(s):  
Steffen Koschmieder ◽  
Francesco D′Alo′ ◽  
Hanna Radomska ◽  
Susumu Kobayashi ◽  
Elena Levantini ◽  
...  
Keyword(s):  
De Novo ◽  
Binding Protein ◽  
Northern Blotting ◽  
Granulocytic Differentiation ◽  
Protein Levels ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Eif2 Alpha

Abstract The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel antineoplastic drug which induces apoptosis of a wide variety of tumor cells in vitro and in vivo and leads to granulocytic differentiation of hematopoietic progenitor cells. We studied the effect of CDDO on CCAAT enhancer binding protein alpha (CEBPA), a transcription factor which is critical for granulocytic differentiation. In HL60 myeloblastic cells, CDDO (0.01 to 2 uM) dose-dependently decreased the number of cells in culture and increased the fraction of apoptotic cells. However, at doses which did not induce apoptosis, CDDO increased the number of granulocytic cells, as assessed by morphology, NBT assay, and FACS, and Northern blotting showed an increase of GCSFR and a decrease of c-myc mRNA. Phagocytosis of FITC-labeled E. coli bacteria by these cells was enhanced by CDDO. While CEBPA mRNA was decreased, CEBPA protein was significantly increased within 24 hours of treatment, and this was not abrogated by preincubation with the caspase inhibitor Z-DEVD-fmk, again suggesting that these effects were independent of apoptosis. CDDO increased the ratio of the transcriptionally active isoform p42 and the inactive p30 isoform 3-fold, and gel shift assays showed enhanced DNA binding to a GCSFR promoter probe. Since eukaryotic translation initiation factors (eIF) have been described to alter the CEBPA protein isoform ratio, we studied the effects of CDDO on eiF2 alpha and eiF4E activity. CDDO increased the phosphorylation of eIF4E and decreased the phosphorylation of eIF2 alpha within 5 hours of treatment, and this was associated with an increase of the p42/p30 CEBPA ratio. In the presence of the translation inhibitor cycloheximide, CEBPA protein levels decreased after 2 hours, suggesting that CDDO did not stabilize CEBPA and that de novo protein synthesis was required for the observed effects. The effect of CDDO on the p42/p30 ratio was mimicked by 2-AP, which inhibits eIF2 alpha phosphorylation, but was independent of PPARgamma and TGFß pathways, as demonstrated by preincubation with GW9662, or TGFß1, respectively. In primary blasts from patients with acute myeloid leukemia (AML), the p42/p30 ratio of CEBPA was enhanced by CDDO treatment. In conclusion, CDDO leads to granulocytic differentiation and translational induction of CEBPA protein. Since CEBPA function is impaired in many patients with AML, CDDO may provide a novel treatment approach for these patients.

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