Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A molecular model of a mammalian membrane-bound cytochrome P450, rat P450 2B1, was constructed in order to elucidate its mode of attachment to the endoplasmic reticulum and the structural basis of substrate specificity. The model was primarily derived from the structure of P450BM-3, which as a class II P450 is the most functionally similar P450 of known structure. However, model development was also guided by the conserved core regions of P450cam and P450terp. To optimally align the P450 2B1 and P450BM-3 sequences, multiple alignment was performed using sequences of five P450s in the II family, followed by minor adjustments on the basis of secondary structure predictions. The resulting P450 2B1 homology model structure was refined by molecular dynamics heating, equilibration, simulation, and energy minimization. The model suggests that the F-G loop serves as both a hydrophobic membrane anchor and entrance channel for hydrophobic substrates from the membrane to the P450 active site. To assess the mode of substrate binding, benzphetamine, testosterone, and benzo[a]pyrene were docked into the active site. The hydrophobic substrate-binding pocket is consistent with the preferences of this P450 toward hydrophobic substrates, while the presence of an acidic Glu-105 in this pocket is consistent with the preference of this P450 for the cationic substrate benzphetamine. This model is thus consistent with several known experimental properties of this P450, such as membrane attachment and substrate selectivity.
 ...
PMID:Molecular modeling of cytochrome P450 2B1: mode of membrane insertion and substrate specificity. 953 74

We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P450 2C9. The chimeric protein shows a reduced CO-difference spectrum absorption at 446 nm, and circular dichroism spectra indicate that the protein is globular. The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin reductase. This chimera provides a novel method for addressing structure-function issues and may prove useful in the design of oxidants for benign and stereospecific synthesis, as well as catalysts for bioremediation of polluted areas. Furthermore, these results provide the first evidence that bacterial P450 enzymes and mammalian P450 enzymes are likely to share a common tertiary structure.
 ...
PMID:Design of a novel P450: a functional bacterial-human cytochrome P450 chimera. 963 25

Steroidogenic acute regulatory (StAR) protein is synthesized in response to tropic hormones to facilitate cholesterol transport to the inner mitochondrial membrane-bound P450 side-chain cleavage enzyme (P450scc), the first enzymatic step in the steroid hormone biosynthetic pathway. Gonadotropins activate expression of their target genes via the cAMP second messenger system. We have demonstrated that cAMP administration to rat luteal cells stimulates expression of both StAR messenger RNA and protein. Because cholesterol delivery is the first regulated step in steroidogenesis, and because StAR messenger RNA levels are increased in response to tropic hormone and cAMP stimulation, the mechanism by which tropic hormones/cAMP stimulate transcription needs to be elucidated. To this end, approximately 2.7 kb of the rat StAR promoter was isolated and sequenced. Sequence analysis revealed the presence of a TATA-like element as well as multiple regulatory motifs including steroidogenic factor 1 (SF-1) binding sites, an estrogen receptor half-site, and two AP-1 sites within the promoter region. 5'-RACE experiments determined the transcription start site to be located 82 bp upstream of the ATG translation start codon. Electrophoretic mobility shift assays and supershift analysis demonstrated SF-1 binding to three SF-1 binding sites in the rat StAR promoter with high affinity and two SF-1 binding sites with low affinity. Transfection of mouse Y1 adrenal tumor cells and human bladder carcinoma cells (HTB9s) with the rat StAR promoter demonstrated that SF-1 was able to activate transcription of the luciferase reporter gene and that the rat StAR promoter was responsive to cAMP. Nested deletions of the rat StAR promoter (1.9 kb) identified a region between -1413 and -998 that is essential for maximal activation of the rat StAR gene in HTB9 cells; however, deletion of this region does not affect responsiveness to cAMP. 5'-Deletion and site-directed mutagenesis experiments demonstrated that the SF-1 motifs identified within the rat StAR promoter (located at positions -764, -455, and -106) were sufficient to activate transcription as well as confer cAMP responsiveness to the rat StAR gene. Site-directed mutagenesis studies using the smallest promoter fragment demonstrated that the two proximal SF-1 binding sites are crucial for StAR gene transcription, both at a basal level and in response to cAMP stimulation. These studies provide novel insights into the regulation of the rat StAR gene at the transcriptional level by SF-1.
 ...
PMID:Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1. 983 18

Some xenobiotics induce membrane-bound drug metabolizing enzymes (Xme) and a profound proliferation of the endoplasmic reticulum (ER) in vivo. However these effects are much weaker in vitro, possibly due to absence of certain transcription factors. We tested the possibility that ER proliferation can affect the level of ER-resident enzymes even in the absence of transcriptional activation. For this purpose we analysed the effects of compactin, which has been shown to induce ER proliferation in vitro, on recombinant Xme, which were expressed from a constitutive viral promoter. High levels of recombinant UDP-glucuronosyltransferase UGT1A6 were achieved by amplification of the UGT1A6 cDNA using the dihydrofolate reductase cDNA as selectable marker in DHFR- CHO cells. Treatment of the resulting cell lines with lipoprotein-deficient serum in the absence and presence of compactin for 5 days resulted in a 1.3- and 2.3-fold, respectively, increase of the UGT enzyme activity towards 4-methylumbelliferone, paralleled by an induction of immunoreactive UGT1A6 protein. Similarly, treatment with this 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor increased the endogenous P450 reductase activity 2.6-fold, concomitant with an increase of immunodetectable protein. As expected compactin induced the level of 3-hydroxy-3-methylglutaryl-CoA reductase. Increased levels of this protein have been associated with a proliferation of the ER. Compactin treatment of a separate cell line that expressed recombinant human P450 reductase increased this enzyme activity fivefold. Pulse-chase experiments revealed that the induction of the recombinant Xme by compactin was most likely due to decreased protein degradation. Our results show that enzyme systems unrelated to those involved in cholesterol biosynthesis are affected by compounds known to affect membrane biogenesis. Since this effect extends to heterologously expressed enzymes, it also provides an efficient means by which to increase the levels of recombinant ER proteins.
 ...
PMID:Treatment of mammalian cells with the endoplasmic reticulum-proliferator compactin strongly induces recombinant and endogenous xenobiotic metabolizing enzymes and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro. 991 63

The pale-brown chafer, Phyllopertha diversa, utilizes an unusual alkaloid, 1,3-dimethyl-2,4-(1H,3H)-quinazolinedione, as its sex pheromone. This compound is rapidly degraded in vitro by the antennal protein extracts from this scarab beetle. Demethylation at the N-1 position and hydroxylation of the aromatic ring have been identified as the major catabolic pathways. The enzyme responsible for the pheromone degradation is membrane-bound, requires NAD(P)H for activity and is sensitive to cytochrome P450 inhibitors, such as proadifen and metyrapone. The ability to metabolize this unusual pheromone was not detected in 12 species tested, indicating that the P450 system, specific to male P. diversa antennae, has evolved as a mechanism for olfactory signal inactivation.
 ...
PMID:Degradation of an alkaloid pheromone from the pale-brown chafer, Phyllopertha diversa (Coleoptera: Scarabaeidae), by an insect olfactory cytochrome P450. 1057 Sep 35

Using the mRNA differential display technique and Western blot analysis, the present study demonstrates that induction of KAR2 occurs when misfolded membrane-bound cytochrome P450, mutated in its cytosolically exposed domain, is expressed in Saccharomyces cerevisiae. Using various KAR2 promoter constructs in front of the Escherichia coli beta-galactosidase reporter gene, we found a fast and strong induction through the heat shock element (HSE), which was enhanced several fold by its adjacent GC-rich region. Additionally, a less pronounced induction was detected for the UPR element (UPRE). As expected, this response was absent in the ire1 disruptant strain. However, the HSE-mediated induction was enhanced upon disruption of IRE1 suggesting that the HSE pathway can compensate for the lack of a functional UPR pathway. Western blotting confirmed that Kar2p levels were increased to the same extent in the ire1 disruptant and in the non-disruptant strain. Removal of the P450 membrane-spanning region also abolished the UPRE-mediated induction of KAR2 transcription, but the HSE-mediated response remained. The data show for the first time that the transcription of KAR2 is significantly induced in response to a misfolded membrane-bound endoplasmic reticulum protein, and identifies the HSE and UPRE regions as KAR2 promoter elements responding to the misfolded cytosolic P450 domain and to the membrane-integrated mutant P450, respectively.
 ...
PMID:Misfolded membrane-bound cytochrome P450 activates KAR2 induction through two distinct mechanisms. 1057 60

The term "cytochrome P450" first appeared in literature in 1962. It was a microsomal membrane-bound hemoprotein without known physiological functions at that time and was characterized by a unique 450-nm optical absorption peak of its carbon monoxide-bound form, which was originally reported as the spectrum of a novel "microsomal carbon monoxide-binding pigment" in 1958. Elucidation of its function as the oxygenase in 1963 triggered a rapid expansion of research on this hemoprotein. Annual numbers of the published papers dealing with cytochrome P450, which were listed in Biological Abstracts, increased from 60 in 1970 to 500 in 1980, 900 in 1990, and 1500 in 1997. Cytochrome P450 is now regarded as the collective name of a large family of hemoproteins, "cytochrome P450 superfamily, "which seems to have diversified from a single ancestral protein to many forms during the course of biological evolution and is distributed widely among various forms of life from animals and plants to fungi and bacteria. Multicellular eukaryotic organisms including animals and plants have about 100 or more P450 genes in their genomes, and those many P450 genes are expressed tissue specifically and developmental stage specifically, indicating their diverse physiological functions. In mammals, various P450s participate in the biosynthesis and metabolism of sterols and steroid hormones and the metabolism of various lipid biofactors including eicosanoids, vitamin D3, and retinoids. Oxidative metabolism of foreign hydrophobic compounds as the first step of their excretion from the animal body is apparently another major function of cytochrome P450, which protects animals from noxious foreign compounds, man-created and natural.
 ...
PMID:Forty years of cytochrome P450. 1060 7

Deletion of the N-terminal membrane-spanning domain from microsomal P450s 2C5 and 2C3 generates the enzymes, 2C5dH and 2C3dH, that exhibit a salt-dependent association with membranes indicating that they retain a monofacial membrane interaction domain. The two proteins are tetramers and dimers, respectively, in high salt buffers, and only 2C5dH requires phospholipids to reconstitute fully the catalytic activity of the enzyme. Amino acid residues derived from P450 2C3dH between residues 201 and 210 were substituted for the corresponding residues in P450 2C5 to identify those that would diminish the membrane interaction, the phospholipid dependence of catalysis, and aggregation of 2C5dH. Each of four substitutions, N202H, I207L, S209G, and S210T, diminished the aggregation of P450 2C5dH and produced a monomeric enzyme. The N202H and I207L mutations also diminished the stimulation of catalytic activity by phospholipid and reduced the binding of P450 2C5dH to phospholipid vesicles. The modified enzymes exhibit rates of progesterone 21-hydroxylation that are similar to that of P450 2C5dH. These conditionally membrane-bound P450s with improved solubility in high salt buffers are suitable for crystallization and structural determination by x-ray diffraction studies.
 ...
PMID:Engineering microsomal cytochrome P450 2C5 to be a soluble, monomeric enzyme. Mutations that alter aggregation, phospholipid dependence of catalysis, and membrane binding. 1064 12

In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B 1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116-134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.
 ...
PMID:Synthetic peptide mimics of a predicted topographical interaction surface: the cytochrome P450 2B1 recognition domain for NADPH-cytochrome P450 reductase. 1088 69

Expression of the membrane-bound cytochrome P450 2B4 by the pLW01-P450 expression vector, which utilizes a T7 promoter, is markedly improved by employing Escherichia coli strain C41(DE3) [Miroux, B., and Walker, J. (1996) J. Mol. Biol 260, 289--298; Bridges, A., Gruenke, L., Chang, Y.-T., Vasker, I., Loew, G., and Waskell, L. (1998) J. Biol. Chem. 273, 17036--17049]. Using this expression system, it was possible to routinely obtain an average of 50--60 mg and as high as 100 mg of cyt P450 2B4 per liter of cell culture in volumes of 500 ml. An improved purification procedure for cyt P450 2B4 is also described which allows recovery of 30% of the expressed protein. It was possible in one step using B-PER reagent and polyoxyethylene-9-lauryl ether to both lyse the E. coli and solubilize the expressed cyt P450. Cyt P450 2B4 with a specific content of 17 nmol/mg protein and a single band on polyacrylamide gel electrophoresis was routinely isolated. The yield of cyt P450 from the improved purification procedure is twice that from the original procedure and the purity of the recovered protein typically has a specific content of 17 nmol cyt P450/mg of protein.
 ...
PMID:Overexpression and purification of the membrane-bound cytochrome P450 2B4. 1123 92


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>