Development of CYB5-fusion monitoring system for efficient periplasmic expression of multimeric proteins in Escherichia coli.

Despite all advances of heterologous expression of recombinant proteins in Escherichia coli, expression of multidomain disulphide-rich proteins faces some problems due to the absence of the possibility to monitor the process in real-time. Here we provide a CYB5-fusion system - cytochrome b5 fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. We validated this system by Fab and scFv antibody fragments expression in order to improve antibody-derived molecules characterization and to simplify their usage. The combination of redox dependent absorbance spectrum of red-colored cytochrome b5 with its high value molar extinction coefficient allows us to monitor antibody fusion proteins localization, redox state and quantify them in reliable way in turbid solutions. Moreover, it was revealed that due to outstanding stability and solubility, cytochrome b5 significantly enhances expression level of Fab/scFv antibody fragments in Escherichia coli periplasm.

CYB5D2 displays tumor suppression activities towards cervical cancer.

Cervical cancer is caused by infections with human papillomaviruses (HPV) and genetic alternations in the cervical epithelium. While the former is well studied, the latter remains unclear. We report here that CYB5D2/Neuferricin possesses tumor suppressing activity towards cervical tumorigenesis. Ectopic expression of CYB5D2 did not affect HeLa cell proliferation and the cell's ability to form xenograft tumors, but significantly inhibited HeLa cell invasion in vitro and the cell-produced lung metastasis in NOD/SCID mice. Knockdown of CYB5D2 enhanced HeLa cell invasion. Two mutations in CYB5D2, the substitutions of arginine (R) 7 with either proline (P) or glycine (G), were reported in colon cancer. Both CYB5D2(R7P) and CYB5D2(R7G) were incapable of inhibiting HeLa cell invasion. CYB5D2 binds heme, in which aspartate (D) 86 is required. While CYB5D2(D86G) is heme-binding defective, it inhibited HeLa cell invasion. On the other hand, CYB5D2(R7P) and CYB5D2(R7G) bound heme but did not inhibit HeLa cell invasion. Collectively, CYB5D2 inhibits HeLa cell invasion independently of its heme binding. Furthermore, immunohistochemistry examination of CYB5D2 expression in 20 normal cervical tissues and 40 cervical squamous cell carcinomas (SCC) revealed a CYB5D2 reduction in 87.5% (35/40) of SCC. Analysis of CYB5D2 gene expression and genomic alteration data available from Oncomeine™ detected significant reductions of CYB5D2 mRNA in 40 SCCs and CYB5D2 gene copy number in 107 SCCs. Collectively, we provide evidence that CYB5D2 is a candidate tumor suppressor of cervical tumorigenesis.

Development of adrenal cortical zonation and expression of key elements of adrenal androgen production in the chimpanzee (Pan troglodytes) from birth to adulthood.

The basis for the pattern of adrenal androgen production in the chimpanzee, which resembles that of humans, is poorly defined. We characterized the developmental zonation and expression of elements of the androgen biosynthetic pathway in the chimpanzee adrenal. The newborn adrenal contained a broad fetal zone (FZ) expressing CYP17, SULT2A1, and Cytochrome B5 (CB5) but not HSD3B; the outer cortex expressed HSD3B but not SULT2A1 or CB5. During infancy, the FZ involuted and the HSD3B-expressing outer cortex broadened. By 3years of age, a thin layer of cells that expressed CB5, SULT2A1, and CYP17 adjoined the medulla and likely represented the zona reticularis; the outer cortex consisted of distinct zonae fasiculata and glomerulosa. Thereafter, the zona reticularis broadened as also occurs in the human. The adult chimpanzee adrenal displayed other human-like characteristics: intramedullary clusters of reticularis-like cells and also a cortical cuff of zona fasiculata-like cells adjoining the central vein.

Co-expression of human cytochrome b5 increases expression of cytochrome P450 3A4 in Escherichia coli by stabilizing mRNA.

CYP3A4 is the most abundant cytochrome P450 in the human liver. The expression level of CYP3A4 when coexpressed with cytochrome b(5) (cyt b(5)) in Escherichia coli was 20-60% higher than that when it was expressed alone over an extended period (48-72 h). This time-dependent elevation in coexpression with cyt b(5) was a result of an increase in CYP3A4 mRNA half-life; no significant change in CYP3A4 degradation was seen in the bacterial protease fraction. These results suggest that the higher CYP3A4 levels observed upon coexpression with cyt b(5) primarily resulted from CYP3A4 mRNA stabilization by cyt b(5).

[Optimization of tri-expression of human CYP3A4 with POR and cyt b5 in Sf 9 cells].

OBJECTIVE: To investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells. METHODS: The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. RESULTS: When the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 µmol/L,0.52 µmol/(min*g protein) and 4.34 ml/(min*g protein), respectively. CONCLUSION: The conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

A biochemical analysis of the constraints of tail-anchored protein biogenesis.

TA (tail-anchored) proteins utilize distinct biosynthetic pathways, including TRC40 (transmembrane domain recognition complex of 40 kDa)-mediated, chaperone-dependent and/or unassisted routes to the ER (endoplasmic reticulum) membrane. We have addressed the flexibility of cytosolic components participating in these pathways, and explored the thermodynamic constraints of their membrane insertion, by exploiting recombinant forms of Sec61ß and Cytb5 (cytochrome b5) bearing covalent modifications within their TA region. In both cases, efficient membrane insertion relied on cytosolic factors capable of accommodating a surprising range of covalent modifications to the TA region. For Sec61ß, we found that both SGTA (small glutamine-rich tetratricopeptide repeat-containing protein α) and TRC40 can bind this substrate with a singly PEGylated TA region. However, by introducing two PEG [poly(ethylene glycol)] moieties, TRC40 binding can be prevented, resulting in a block of subsequent membrane integration. Although TRC40 can bind Sec61ß polypeptides singly PEGylated at different locations, membrane insertion is more sensitive to the precise location of PEG attachment. Modelling and experimentation indicate that this post-TRC40 effect results from an increased energetic cost of inserting different PEGylated TA regions into the lipid bilayer. We therefore propose that the membrane integration of TA proteins delivered via TRC40 is strongly dependent upon underlying thermodynamics, and speculate that their insertion is via a phospholipid-mediated process.

Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells.

Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b(5) (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested [Kimura, S., et al. (2005) J. Biochem. 137, 523-533]. In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD sequence increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation but also for increasing the soluble form of recombinant proteins in E. coli cells.

Phenotype of hepatic xenobiotic metabolizing enzymes and CYP450 isoforms of sanguinarine treated rats: effect of P450 inducers on its toxicity.

Catalytic and immunochemical activities of cytochrome P450 (CYP) isoforms were investigated in argemone alkaloid, sanguinarine (SAN) intoxicated rats, pre-treated with different CYP inducers. SAN treated control (CON) and ethanol (ET), 3- methylcholantherene (MC) or dexamethasone (DEX) pre-exposed rats, resulted in 48, 64, 47 and 33% decrease in CYP content. SAN exposure to CON, and DEX, MC or ET pre-treated animals caused a decrease (22-37%) in glutathione-S-transferase (GST) activity, however, quinone reductase (QR) activity decreased (26-45%) in the MC pre-exposed group. Similarly, western-blot analysis of hepatic CYP1A1 and CYP1A2 showed a decrease (27-37%) in MC pre-treated SAN exposed animals. Further, a decrease in mortality in the SAN+MC (25%) group compared to SAN treated animals was also observed. The results suggest that inhibition of CYP 1A1, 1A2, 2D1, 2E1, 3A1, and Phase II enzymes by SAN augments its toxicity, whereas attenuation of SAN toxicity by MC may be due to removal of parent compound/metabolites from the body.

Cytochrome b5 expression in gonadectomy-induced adrenocortical neoplasms of the domestic ferret (Mustela putorius furo).

Whereas the adrenal glands of healthy ferrets produce only limited amounts of androgenic steroids, adrenocortical neoplasms that arise in neutered ferrets typically secrete androgens or their derivative, estrogen. The 17,20-lyase activity of cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) must increase to permit androgen biosynthesis in neoplastic adrenal tissue. We screened ferret adrenocortical tumor specimens for expression of cytochrome b(5) (cyt b(5)), an allosteric regulator that selectively enhances the 17,20-lyase activity of P450c17. Cyt b(5) immunoreactivity was evident in 24 of 25 (96%) adrenocortical adenomas/carcinomas from ferrets with signs of ectopic sex steroid production. Normal adrenocortical cells lacked cyt b(5), which may account for the low production of adrenal androgens in healthy ferrets. Other markers characteristic of gonadal somatic cells, such as luteinizing hormone receptor, aromatase, and GATA4, were coexpressed with cyt b(5) in some of the tumors. We concluded that cyt b(5) is upregulated during gonadectomy-induced adrenocortical neoplasia and is a marker of androgen synthetic potential in these tumors.

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles.

A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3mg per g of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.

Regulation of the adrenal androgen biosynthesis.

The human adrenal reticularis produces the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S). As opposed to the cortisol and aldosterone little is known regarding the mechanisms that regulate the production of the adrenal androgens. Several recent studies have shown that type II 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5 (CYB5), and steroid sulfotransferase (SULT2A1) play an important role in the regulation of adrenal androgen production. Specifically, adrenal production of DHEA-S is correlated with reticularis expression of SULT2A1 and CYB5. In contrast, HSD3B2 has an inverse correlation with adrenal androgen production likely due to its unique ability to remove precursors from the pathway leading to DHEA. Therefore, its expression is limited to the adrenal glomerulosa/fasciculata but not in reticularis. The differential expression of these three proteins appears to be critical for reticularis function. In this review, we focus on studies that have begun to define the mechanisms regulating the transcription of these genes. Understanding the mechanisms controlling differential expression of these proteins should provide novel information about the human adrenal reticularis and its production of DHEA and DHEA-S.

Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis.

We investigated the direct constitution of membrane proteins into giant liposomes in cell-free (in vitro) protein synthesis. Giant liposomes were present in a translation reaction cocktail of a wheat germ cell-free protein translation system. Apo cytochrome b(5) (b5) and its fusion proteins were synthesized and directly localized in the liposomes. After the translation reaction, the proteo-liposomes were isolated by simplified discontinuous density-gradient centrifugation. Apo cytochrome b(5) conjugated dihydrofolate reductase (DHFR) was synthesized in the same procedure and the protein was directly displayed on the liposome surface. b5 acts as a "hydrophobic tag" for recruitment to the liposome surface.

Discovery and characterization of a cytochrome b5 variant in humans with impaired hydroxylamine reduction capacity.

OBJECTIVES: We have shown that cytochrome b5 (cyt b5), along with its reductase, NADH cytochrome b5 reductase (b5R), is capable of direct xenobiotic biotransformation. We hypothesized that functionally significant genetic variability in cyt b5 could be found in healthy individuals. BASIC METHODS: Cyt b5 cDNAs were prepared from peripheral blood mononuclear cells from 63 individuals. MAIN RESULTS: One individual was heterozygous for a sequence variant in cyt b5 (A178G), with a predicted amino acid substitution of T60A. This variant, when expressed in Escherichia. coli, maintained a similar Vmax for the hydroxylamines of sulfamethoxazole, 4-aminobiphenyl, and 2-amino-l-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), compared with wild type cyt b5, with a modestly increased Km (2 to 3.5-fold) for each substrate. When expressed in a mammalian system (HeLa cells), however, T60A was associated with a 70% reduction in cyt b5 protein expression compared with wild type. mRNA expression for both isoforms were comparable in HeLa cells, and translation of these mRNAs in a rabbit reticulocyte lysate system with inhibited proteasomal machinery were also similar. Incubation of these translated enzymes with uninhibited rabbit reticulocyte lysate, however, indicated greater susceptibility of T60A to proteasomal degradation. CONCLUSIONS: These data indicate that a naturally occurring variant in cyt b5, T60A, leads to modestly altered affinity for hydroxylamine substrates and dramatically reduced cyt b5 expression. Work is underway to determine the prevalence of this and other variants in cyt b5 or b5R in a larger population, and to determine the association of such variants with differences in hydroxylamine reduction in vivo.

Subcellular/tissue distribution and responses to oil exposure of the cytochrome P450-dependent monooxygenase system and glutathione S-transferase in freshwater prawns (Macrobrachium malcolmsonii, M. lamarrei lamarrei).

Subcellular fractions (mitochondrial, cytosolic and microsomal) prepared from the tissues (hepatopancreas, muscle and gill) of freshwater prawns Macrobrachium malcolmsonii and Macrobrachium lamarrei lamarrei were scrutinized to investigate the presence of mixed function oxygenase (MFO) and conjugating enzymes (glutathione-S-transferase, GST). Cytochrome P450 (CYP) and other components (cytochrome b(5); NADPH-cytochrome c (CYP) reductase and NADH-cytochrome c-reductase activities) of the MFO system were predominantly present in the hepatic microsomal fraction of M. malcolmsonii and M. lamarrei lamarrei. The results are in agreement with the notion that monooxygenase system is mainly membrane bound in the endoplasmic reticulum, and that the hepatopancreas is the major metabolic tissue for production of biotransformation enzymes in crustaceans. Further, the prawns were exposed to two sublethal (0.9 ppt (parts per thousand) and 2.3 ppt) concentrations of oil effluent. At the end of 30th day, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. The accumulations of hydrocarbon in the tissues gradually increased when exposed to sublethal concentrations of oil effluent and were associated with significantly enhanced levels of cytochrome P450 (180.6+/-6.34 pmol mg(-1) protein (P<0.05 versus control, 136.5+/-7.1 pmol mg(-1) protein) for 2.3 ppt and 305.6+/-8.5 pmol mg(-1) protein (P<0.001 versus control, 132.3+/-6.8 pmol mg(-1) protein] for 0.9 ppt of oil exposed M. malcolmsonii; 150+/-6.5 pmol mg(-1 )protein (P<0.01 versus control, 84.6+/-5.2 pmol mg(-1) protein) for 2.3 ppt and 175+/-5.5 pmol mg(-1) protein (P<0.01 versus control, 87.6+/-5.4 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei), NADPH cytochrome c-reductase activity (14.7+/-0.6 nmol min(-1 )mg(-1) protein (P<0.05 versus control, 6.8+/-0.55 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 12.1+/-0.45 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 6.9+/-0.42 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. malcolmsonii; 12.5+/-0.31 nmol min(-1 )mg(-1) protein (P<0.001 versus control, 4.6+/-0.45 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 9.6+/-0.32 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 4.9+/-0.41 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei) and cytochrome b(5 )(124.8+/-3.73 pmol mg(-1) protein (P<0.01 versus control, 76.8+/-4.2 pmol mg(-1) protein) for 2.3 ppt and 115.3+/-3.86 pmol mg(-1) protein (P<0.01 versus control, 76.4+/-4.25 pmol mg(-1 )protein) for 0.9 ppt of oil exposed M. malcolmsonii and 110+/-3.11 pmol mg(-1) protein (P<0.01 versus control, 63.7+/-3.24 pmol mg(-1 )protein) for 2.3 ppt and 95.3+/-2.63 pmol mg(-1) protein (P<0.01 versus control, 61.4+/-2.82 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei). The enhanced levels of biotransformation enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in crustaceans, and the response offers the possibility of use as a biomarker for the early detection of oil pollution.

Using human microarrays to identify differentially expressed genes associated with increased steroidogenesis in boars.

Human microarrays are readily available, and it would be advantageous if they could be used to study gene expression in other species, such as pigs. The objectives of this research were to validate the use of human microarrays in the analysis of porcine gene expression, to assess the variability of the data generated, and to compare gene expression in boars with different levels of steroidogenesis. Cytochrome b5 (CYB5) expression was used to assess array detection sensitivity. Samples having high or low CYB5 RNA levels were hybridized to microarrays to determine if the known expression difference could be detected. Six hybridizations were conducted using human microarrays containing 3840 total spots representing 1718 characterized human ESTs. To analyze gene expression in boars with different levels of steroidogenesis, testis RNA from four boars with high levels of plasma estrone sulphate was hybridized to testis RNA from four boars with lower levels. Eight microarray hybridizations were conducted including fluor-flips. Self-self hybridizations were also conducted to assess the variability of array experiments. The Cy5 and Cy3 intensity values for each array were normalized using a locally weighted linear regression (LOESS). Statistical significance was assessed using a Student's t-test followed by the Benjamini and Hochberg multiple testing correction procedure. Quantitative real-time PCR (Q-RT-PCR) was used to verify select gene expression differences. The results show that CYB5 was significantly overexpressed in the high CYB5 sample by 1.8 fold (P < 0.05), verifying the known expression difference. The average log2 ratio of the majority of genes (1643) falls within one standard deviation of the mean, indicating the data were reproducible. In the high versus low steroidogenesis experiment, seven genes were significantly overexpressed in the high group (P < 0.05). Quantitative real-time PCR was used to validate five genes with the highest fold change, and the results corroborated those found by the microarray experiments. The results of the self-self hybridizations showed that no genes were significantly differentially expressed following the application of the Benjamini and Hochberg multiple testing correction procedure. The results presented in this report show that human arrays can be used for gene expression analysis in pigs.

Induction of rat hepatic cytochrome P-450 by ketamine and its toxicological implications.

Ketamine is a common intravenous anesthetic and a frequent drug of abuse, alone or in combination with cocaine. However, the pharmacokinetic effects of ketamine have not been fully investigated. This study determined the effects of ketamine on cytochrome P-450 (P-450)-dependent catalytic activities, protein levels, and hepatotoxicity using male Wistar rats treated with 10, 20, 40, or 80 mg/kg ketamine intraperitoneally twice daily for 4 d. Treatment with ketamine produced a dose-dependent increase of pentoxyresorufin O-dealkylation activity of liver microsomes. Treatment with 80 mg/kg ketamine resulted in 14-, 3-, and 2-fold rise in O-dealkylation of pentoxyresorufin, ethoxyresorufin, and methoxyresorufin of rat liver microsomes, respectively. The treatment produced 31% and 86% increases in 7-ethoxycoumarin O-deethylation and erythromycin N-demethylation, respectively. In addition, aniline hydroxylation activity was elevated by 62%. Protein blot analysis of liver microsomal proteins revealed that 80 mg/kg ketamine induced P-450 1A, 2B, 2E1, and 3A proteins by 2-, 13-, 2-, and 2-fold, respectively. In reversibility study, ketamine-induced pentoxyresorufin O-dealkylation, 7-ethoxycoumarin O-deethylation, erythromycin N-demethylation, and methoxyresorufin O-demethylation activities of liver microsomes prepared from rats 4 d after ketamine treatment were 75%, 48%, 29%, and 38% lower than the respective activities of liver microsomes prepared from rats 1 d after treatment. Protein blot analysis showed that ketamine-induced P-450 2B1/2 proteins also decreased in a time-dependent manner in 4 d. In hepatotoxicity study, treatment of rats with 1 ml/kg CCl4 produced a 7-fold increase in serum alanine aminotransferase activity level and a 17-fold rise in rats pretreated with 80 mg/kg ketamine for 4 d. Treatment of ICR mice with 120 mg/kg cocaine produced a 17% mortality, whereas the same dose of cocaine produced a 50% mortality in mice pretreated with ketamine. Treatment of mice with 100 mg/kg cocaine produced a 76-fold increase in serum alanine aminotransferase activity level and a 260-fold rise in mice pretreated with 80 mg/kg ketamine for 4 d. The present study shows that ketamine induces the expression of multiple forms of P-450 in rat liver microsomes and increases CCl4-induced liver toxicity and cocaine-mediated acute toxicity. Other potential pharmacological or toxicological events related to ketamine use need to be further explored.

Regulation of cytochrome b5 gene transcription by Sp3, GATA-6, and steroidogenic factor 1 in human adrenal NCI-H295A cells.

Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances.

High level expression of peptides and proteins using cytochrome b5 as a fusion host.

A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa).

Bioengineering and characterization of DNA-protein assemblies floating on supported membranes.

This chapter describes the design, practical construction, and characterization of P-DNA and their applications in building a new generation of DNA chips. P-DNAs are artificial covalent assemblies involving a histidine tag head able to bind to modified phospholipids, a core protein domain derived from cytochrome b5 by genetic engineering that features specific spectroscopic and electrochemical properties useful for detection, a synthetic linker acting as a spacer, and an oligonucleotide acting as a probe. P-DNA has the property of being able to efficiently self-associate to a supported bilayer including nickel-iminodiacetate-modified phospholipids. The construction of P-DNA and its interaction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using a Biacore system or equivalent. P-DNA chips feature unique properties including tunable surface density of probes; very low nonspecific interaction with external DNA; lateral mobility, minimizing-steric interaction; optimization of hybridization efficiency; and, potentially, recognition by multiple probes of a single target and perfectly defined and homogeneous structure, permitting high density up to a compact monolayer. Potential applications of this new device are multiple, including high-sensitivity and high-selectivity chips for DNA-DNA, DNA-RNA, or DNA-protein interactions.

Evidence of significant contribution from CYP3A5 to hepatic drug metabolism.

CYP3A4 and CYP3A5 exhibit significant overlap in substrate specificity but can differ in product regioselectivity and formation activity. To further explore this issue, we compared the kinetics of product formation for eight different substrates, using heterologously expressed CYP3A4 and CYP3A5 and phenotyped human liver microsomes. Both enzymes displayed allosteric behavior toward six of the substrates. When it occurred, the "maximal" intrinsic clearance was used for quantitative comparisons. Based on this parameter, CYP3A5 was more active than CYP3A4 in catalyzing total midazolam hydroxylation (3-fold) and lidocaine demethylation (1.4-fold). CYP3A5 exhibited comparable metabolic activity as CYP3A4 (90-110%) toward dextromethorphan N-demethylation and carbamazepine epoxidation. CYP3A5-catalyzed erythromycin N-demethylation, total flunitrazepam hydroxylation, testosterone 6beta-hydroxylation, and terfenadine alcohol formation occurred with an intrinsic clearance that was less than 65% that of CYP3A4. Using two sets of human liver microsomes with equivalent CYP3A4-specific content but markedly different CYP3A5 content (group 1, predominantly CYP3A4; group 2, CYP3A4 + CYP3A5), we assessed the contribution of CYP3A5 to product formation rates determined at low substrate concentrations (< or = Km). Mean product formation rates for group 2 microsomes were 1.4- to 2.2-fold higher than those of group 1 (p < 0.05 for 5 of 8 substrates). After adjusting for CYP3A4 activity (itraconazole hydroxylation), mean product formation rates for group 2 microsomes were still significantly higher than those of group 1 (p < 0.05 for 3 substrates). We suggest that, under conditions when CYP3A5 content represents a significant fraction of the total hepatic CYP3A pool, the contribution of CYP3A5 to the clearance of some drugs may be an important source of interindividual variability.