Fluorescence Study Of A Mutant Cytochrome-b5 With A Single Tryptophan In The Membrane-binding Domain
b5; Biochemistry & Molecular Biology; energy; indole; mutagenesis; probes; residue; Spectroscopy; vesicles
Fluorescence studies of cytochrome b5 are complicated by the presence of three tryptophans, at positions 108, 109, and 112, in the membrane-binding domain. The cDNA for rabbit liver cytochrome b5, isolated from a lambda-gt11 library, was used to generate a mutated mRNA where the codons for tryptophans-108 and -112 were replaced by codons for leucine. The sequence was expressed in Escherichia coli and the mutant protein was isolated. This mutant protein had the expected absorption spectrum, and its amino acid composition was confirmed by amino acid analysis and by DNA sequencing of the construct. The fluorescence emission spectrum of the mutant is blue-shifted and is narrower than that of the native protein. The quantum yield of the mutant protein, per molecule, is only 60% of that of the native protein, and the enhancement when bound to lipid vesicles or detergent micelles is higher for the mutant. Fluorescence anisotropy measurements and quenching studies using brominated lipids suggest that the fluorescence of the native protein is due to tryptophans-109 and -108 while tryptophan-112 does not emit but undergoes nonradiative energy transfer to tryptophan-108. With this mutant, it was shown that incomplete energy transfer from tyrosines-126 and -129 to tryptophan-109 occurs when the membrane binding domain is inserted into lipid vesicles, which suggests that the membrane-binding domain does not exist in a tight hairpin loop.
Ladokhin A S; Wang L; Steggles A W; Holloway P W
Biochemistry
1991
1991-10
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1021/bi00106a018" target="_blank" rel="noreferrer noopener">10.1021/bi00106a018</a>
Transcriptional regulation of human oxysterol 7 alpha-hydroxylase gene (CYP7B1) by Sp1.
Humans; Protein Binding; Gene Expression Regulation; Cell Line; Transfection; Base Sequence; Binding Sites/genetics; Molecular Sequence Data; Cytochrome P450 Family 7; Mutagenesis; Luciferases/genetics/metabolism; Recombinant Fusion Proteins/genetics/metabolism; CpG Islands/genetics; Cytochrome P-450 Enzyme System/*genetics/metabolism; DNA/genetics; Sequence Deletion; Sp1 Transcription Factor/metabolism/*physiology; Steroid Hydroxylases/*genetics/metabolism; Cultured; Binding; Competitive; Transcription; Genetic; Enzymologic; Tumor Cells; Site-Directed; Regulatory Sequences; Nucleic Acid/genetics
Oxysterol 7 alpha-hydroxylase catalyzes hydroxylation of oxysterols and neurosterols and plays a role in the alternative bile acid synthesis pathway. This gene is widely expressed in many organs and peripheral tissues and may protect tissues from the toxicity of oxysterols. Mutation in CYP7B1 caused neonatal cholestasis. To examine the regulatory mechanisms governing CYP7B1 expression, the 5' flanking sequence of the CYP7B1 was analyzed and revealed a CpG island of about 1.2 kb. Transient transfection assays of deletion mutants of the CYP7B1 promoter-luciferase reporter gene in human liver-derived HepG2, fibroblast NT1088, and human embryonic kidney 293 cell lines revealed that the region from -291 to +189 was critical for gene transcription. Three GC box sequences located between -25 and +10 were essential for basal transcription because mutations of these sequences markedly reduced promoter activity. Sp1 and Sp3 bound to these sequences as demonstrated by DNase I footprinting assays and electrophoretic mobility shift assay. Thus, regulation of CYP7B1 transcription by Sp1 may play a pivotal role in regulating oxysterol levels, which regulate cholesterol metabolism.
Wu Z; Chiang J Y
Gene
2001
2001-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.
Animals; Rats; Transcription Factors/metabolism; Base Sequence; Molecular Sequence Data; DNA/metabolism; Cholesterol 7-alpha-Hydroxylase/*genetics; Hepatocyte Nuclear Factor 4; Mutagenesis; Luciferases/genetics; Retinoid X Receptors; Phosphoproteins/metabolism; COUP Transcription Factor II; COUP Transcription Factors; *Transcriptional Activation; Bile Acids and Salts/biosynthesis; DNA-Binding Proteins/metabolism; Hormones/*physiology; Oligonucleotide Probes/metabolism; Genes; Cultured; Receptors; Genetic; Cytoplasmic and Nuclear/*physiology; Tumor Cells; Reporter; Retinoic Acid/metabolism; Promoter Regions; Nucleic Acid; Site-Directed; *Receptors; Repetitive Sequences; Steroid
The gene encoding cholesterol 7alpha-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt -149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146- TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139-AGTTCAaggccGGG TAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.
Crestani M; Sadeghpour A; Stroup D; Galli G; Chiang J Y
Journal of lipid research
1998
1998-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Regulation of human sterol 27-hydroxylase gene (CYP27A1) by bile acids and hepatocyte nuclear factor 4alpha (HNF4alpha).
Humans; Cell Line; Transfection; Gene Expression Regulation/drug effects; Base Sequence; Binding Sites/genetics; Response Elements/genetics; Molecular Sequence Data; Mutation; Chenodeoxycholic Acid/pharmacology; Transcription Factors/genetics/*metabolism; Hepatocyte Nuclear Factor 4; Mutagenesis; *DNA-Binding Proteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Bile Acids and Salts/*pharmacology; Cholestanetriol 26-Monooxygenase; DNA/chemistry/genetics; Luciferases/genetics/metabolism; Phosphoproteins/genetics/*metabolism; Recombinant Fusion Proteins/genetics/metabolism; Steroid Hydroxylases/*genetics; DNA; Dose-Response Relationship; Drug; Cultured; Receptors; Tumor Cells; Cloning; Molecular; Sequence Analysis; Promoter Regions; Genetic/*genetics; Cytoplasmic and Nuclear/genetics/metabolism; Site-Directed
Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyses sterol side-chain oxidation of bile acid synthesis from cholesterol, and the first reaction of the acidic bile acid biosynthetic pathway. Hydrophobic bile acids suppress human CYP27A1 gene reporter activity when assayed in human hepatocellular blastoma HepG2 cells. Bile acids also inhibit CYP27A1 reporter activity in human embryonic kidney 293 cells. A putative bile acid response element (BARE) was mapped to a region downstream of nt -147 of the human CYP27A1 gene, within which a binding site for a liver-specific nuclear receptor, HNF4alpha, is identified. HNF4alpha strongly stimulates CYP27A1 gene transcription and mutation of its binding site markedly reduced promoter activity. Results suggest that human CYP27A1 gene transcription is suppressed by bile acids and HNF4alpha plays a pivotal role in transcriptional regulation of this gene.
Chen Wenling; Chiang John Y L
Gene
2003
2003-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Leptin augments recruitment of IRF-1 and CREB to thrombospondin-1 gene promoter in vascular smooth muscle cells in vitro.
*cAMP response element-binding protein; *interferon regulatory factor-1; *leptin; *thrombospondin-1; *transcription; *vascular smooth muscle cells; Binding Sites/genetics; Cells; Chromatin Immunoprecipitation/methods; Cultured; Cyclic AMP Response Element-Binding Protein/*metabolism; Gene Expression Regulation/genetics; Genetic/genetics; Humans; Interferon Regulatory Factor-1/*metabolism; Leptin/*metabolism; Muscle; Mutagenesis; Myocytes; Promoter Regions; Response Elements/genetics; Site-Directed/methods; Smooth; Smooth Muscle/*metabolism; Thrombospondin 1/*genetics/*metabolism; Transcription; Transcriptional Activation/genetics; Transfection/methods; Up-Regulation/genetics; Vascular/*metabolism
We previously reported that high pathophysiological concentrations of leptin, the adipocyte-secreted peptide, upregulate the expression of a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in vascular smooth muscle cells. Moreover, this regulation was found to occur at the level of transcription; however, the underlying molecular mechanisms remain unknown. The goal of the present study was to investigate the specific transcriptional mechanisms that mediate upregulation of TSP-1 expression by leptin. Primary human aortic smooth muscle cell cultures were transiently transfected with different TSP-1 gene (THBS1) promoter-linked luciferase reporter constructs, and luciferase activity in response to leptin (100 ng/ml) was assessed. We identified a long THBS1 promoter (-1270/+750) fragment with specific leptin response elements that are required for increased TSP-1 transcription by leptin. Promoter analyses, protein/DNA array and gel shift assays demonstrated activation and association of transcription factors, interferon regulatory factor-1 (IRF-1) and cAMP response element-binding protein (CREB), to the distal fragment of the THBS1 promoter in response to leptin. Supershift, chromatin immunoprecipitation, and coimmunoprecipitation assays revealed formation of a single complex between IRF-1 and CREB in response to leptin; importantly, recruitment of this complex to the THBS1 promoter mediated leptin-induced TSP-1 transcription. Finally, binding sequence decoy oligomer and site-directed mutagenesis revealed that regulatory elements for both IRF-1 (-1019 to -1016) and CREB (-1198 to -1195), specific to the distal THBS1 promoter, were required for leptin-induced TSP-1 transcription. Taken together, these findings demonstrate that leptin promotes a cooperative association between IRF-1 and CREB on the THBS1 promoter driving TSP-1 transcription in vascular smooth muscle cells.
Sahu Soumyadip; Ganguly Rituparna; Raman Priya
American journal of physiology. Cell physiology
2016
2016-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajpcell.00068.2016" target="_blank" rel="noreferrer noopener">10.1152/ajpcell.00068.2016</a>
Transcriptional regulation of human oxysterol 7alpha-hydroxylase by sterol response element binding protein.
*Transcription Factors; Base Sequence; CCAAT-Enhancer-Binding Proteins/*metabolism; Cytochrome P-450 Enzyme System/*genetics/physiology; Cytochrome P450 Family 7; DNA-Binding Proteins/*metabolism; Enzyme Repression; Genetic; Humans; Molecular Sequence Data; Mutagenesis; Promoter Regions; Response Elements; Sp1 Transcription Factor/antagonists & inhibitors; Steroid Hydroxylases/*genetics/physiology; Sterol Regulatory Element Binding Protein 1; Sterols/metabolism; Transcription
Oxysterol 7alpha-hydroxylase (CYP7B1) metabolizes oxysterols, potent regulators of lipid homeostasis. Very little is known about transcriptional regulation of human CYP7B1. The present results indicate that sterol response element binding protein (SREBP), a family of oxysterol-responsive transcription factors that stimulates cholesterol synthesis, may be an important regulator of CYP7B1. SREBP suppressed a human CYP7B1 luciferase reporter gene in several cell lines, most markedly in rat hepatoma McA-RH7777 cells. An SREBP-1-responsive region was mapped to a GC-rich sequence in the proximal CYP7B1 promoter, containing binding sites for the basal transcriptional activator Sp1. Mutagenesis of this sequence abolished SREBP-1-mediated suppression. Data indicated that SREBP does not bind this sequence but affects the gene indirectly, probably via interaction with Sp1. Our findings indicate that CYP7B1 transcription is controlled by SREBP and reveal a link between oxysterol-sensitive regulators and oxysterol metabolism. We propose that CYP7B1 is important for regulating cellular sterol content and protects against oxysterol-mediated toxicity.
Norlin Maria; Chiang John Y L
Biochemical and biophysical research communications
2004
2004-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.bbrc.2004.02.029" target="_blank" rel="noreferrer noopener">10.1016/j.bbrc.2004.02.029</a>
The opposing effects of retinoic acid and phorbol esters converge to a common response element in the promoter of the rat cholesterol 7 alpha-hydroxylase gene (CYP7A).
*Promoter Regions; Animals; Base Sequence; Cell Line; Cholesterol 7-alpha-Hydroxylase/*genetics/metabolism; DNA Primers; Genetic; Molecular Sequence Data; Mutagenesis; Rats; Signal Transduction; Tetradecanoylphorbol Acetate/*pharmacology; Tretinoin/*pharmacology
The activity of the rat CYP7A/luciferase reporter gene was increased five-fold by all-trans retinoic acid (atRA) or 9-cis retinoic acid (9cRA) in transient transfection assay in HepG2 cells. Cotransfection with retinoid X receptor (RXR) stimulated the promoter activity in the absence of ligand, however, addition of atRA inhibited the transcriptional activity. Cotransfection with retinoic acid receptor (RAR) did not have much effect on CYP7A promoter activity. The CYP7A promoter, when linked upstream to the SV40/ luciferase reporter gene, strongly repressed the phorbol 12-myristate 13-acetate (PMA)-stimulated SV40/ luciferase reporter gene activity. The regions conferring the effects of RA and PMA were mapped to nt-176/ -117 and nt-148/-129, respectively. Several direct repeats of hormone response element (AGTTCA) in this region are required for RA response.
Crestani M; Sadeghpour A; Stroup D; Galli G; Chiang J Y
Biochemical and biophysical research communications
1996
1996-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1006/bbrc.1996.1215" target="_blank" rel="noreferrer noopener">10.1006/bbrc.1996.1215</a>