High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5.

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

Cytochrome b5 reductases: Redox regulators of cell homeostasis.

The cytochrome-b5 reductase (CYB5R) family of flavoproteins is known to regulate reduction-oxidation (redox) balance in cells. The five enzyme members are highly compartmentalized at the subcellular level and function as "redox switches" enabling the reduction of several substrates, such as heme and coenzyme Q. Critical insight into the physiological and pathophysiological significance of CYB5R enzymes has been gleaned from several human genetic variants that cause congenital disease and a broad spectrum of chronic human diseases. Among the CYB5R genetic variants, CYB5R3 is well-characterized and deficiency in expression and activity is associated with type II methemoglobinemia, cancer, neurodegenerative disorders, diabetes, and cardiovascular disease. Importantly, pharmacological and genetic-based strategies are underway to target CYB5R3 to circumvent disease onset and mitigate severity. Despite our knowledge of CYB5R3 in human health and disease, the other reductases in the CYB5R family have been understudied, providing an opportunity to unravel critical function(s) for these enzymes in physiology and disease. In this review, we aim to provide the broad scientific community an up-to-date overview of the molecular, cellular, physiological, and pathophysiological roles of CYB5R proteins.

Regulating Effect of Cytochrome b5 Overexpression on Human Breast Cancer Cells.

Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b5 (Cyt b5) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b5 content. Based on the low content of Cyt b5 in MDA-MB-231 cells, the overexpression of Cyt b5 was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b5 MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H2O2, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b5 most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b5 has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b5 and provides clues for clinical treatment.

Functional variants in cytochrome b5 type A (CYB5A) are enriched in Southwest American Indian individuals and associate with obesity.

OBJECTIVE: This study aimed to identify genetic variants enriched in Southwest American Indian (SWAI) individuals that associate with BMI. METHODS: Whole genome sequencing data (n = 296) were used to identify potentially functional variants that are common in SWAI individuals (minor allele frequency ≥10%) but rare in other ethnic groups (minor allele frequency < 0.1%). Enriched variants were tested for association with BMI in 5,870 SWAI individuals. One variant was studied using a luciferase reporter, and haplotypes that included this variant were analyzed for association with various measures of obesity (n = 917-5,870), 24-hour energy expenditure (24-h EE; n = 419), and skeletal muscle biopsy expression data (n = 207). RESULTS: A 5' untranslated region variant in cytochrome b5 type A (CYB5A), rs548402150, met the enrichment criteria and associated with increased BMI (ß = 2%, p = 0.004). Functionally, rs548402150 decreased luciferase expression by 30% (p = 0.003) and correlated with decreased skeletal muscle CYB5A expression (ß = -0.5 SD, p = 0.0008). Combining rs548402150 with two splicing quantitative trait loci in CYB5A identified a haplotype carried almost exclusively in SWAI individuals that associated with increased BMI (ß = 3%, p = 0.0003) and decreased CYB5A expression, whereas the most common haplotype in all ethnic groups associated with lower BMI and percentage of body fatness, increased 24-h EE, and increased CYB5A expression. CONCLUSIONS: Further studies on the effects of CYB5A on 24-h EE and BMI may provide insights into obesity-related physiology.

Role of cytoglobin in cigarette smoke constituent-induced loss of nitric oxide bioavailability in vascular smooth muscle cells.

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease.

Morphologic and Molecular Characterization of Adrenals and Adrenal Rest Affected by Congenital Adrenal Hyperplasia.

Introduction: Adrenocortical hyperplasia and adrenal rest tumor (ART) formation are common in congenital adrenal hyperplasia (CAH). Although driven by excessive corticotropin, much is unknown regarding the morphology and transformation of these tissues. Our study objective was to characterize CAH-affected adrenals and ART and compare with control adrenal and gonadal tissues. Patients/Methods: CAH adrenals, ART and control tissues were analyzed by histology, immunohistochemistry, and transcriptome sequencing. We investigated protein expression of the ACTH receptor (MC2R), steroidogenic (CYP11B2, CYP11B1, CYB5A) and immune (CD20, CD3, CD68) biomarkers, and delta-like 1 homolog (DLK1), a membrane bound protein broadly expressed in fetal and many endocrine cells. RNA was isolated and gene expression was analyzed by RNA sequencing (RNA-seq) followed by principle component, and unsupervised clustering analyses. Results: Based on immunohistochemistry, CAH adrenals and ART demonstrated increased zona reticularis (ZR)-like CYB5A expression, compared to CYP11B1, and CYP11B2, markers of zona fasciculata and zona glomerulosa respectively. CYP11B2 was mostly absent in CAH adrenals and absent in ART. DLK1 was present in CAH adrenal, ART, and also control adrenal and testis, but was absent in control ovary. Increased expression of adrenocortical marker MC2R, was observed in CAH adrenals compared to control adrenal. Unlike control tissues, significant nodular lymphocytic infiltration was observed in CAH adrenals and ART, with CD20 (B-cell), CD3 (T-cell) and CD68 (macrophage/monocyte) markers of inflammation. RNA-seq data revealed co-expression of adrenal MC2R, and testis-specific INSL3, HSD17B3 in testicular ART indicating the presence of both gonadal and adrenal features, and high expression of DLK1 in ART, CAH adrenals and control adrenal. Principal component analysis indicated that the ART transcriptome was more similar to CAH adrenals and least similar to control testis tissue. Conclusions: CAH-affected adrenal glands and ART have similar expression profiles and morphology, demonstrating increased CYB5A with ZR characteristics and lymphocytic infiltration, suggesting a common origin that is similarly affected by the abnormal hormonal milieu. Immune system modulators may play a role in tumor formation of CAH.

Heme-binding protein CYB5D1 is a radial spoke component required for coordinated ciliary beating.

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

Generation of Arabidopsis lines with a red fluorescent marker for endoplasmic reticulum using a tail-anchored protein cytochrome b5 -B.

The endoplasmic reticulum (ER) is a multifunctional organelle that performs multiple cellular activities in eukaryotes. Visualizing ER using fluorescent proteins is a powerful method of analyzing its dynamics and to understand its functions. However, red fluorescent proteins with both an N-terminal signal peptide (SP) and a C-terminal ER retention tetrapeptide (HDEL) often cause mislocalization to vacuoles or extracellular spaces when they are constitutively expressed in Arabidopsis. To obtain a red fluorescent ER marker, we selected Arabidopsis cytochrome b5 -B (Cb5-B), a tail-anchored (TA) protein on the ER membrane. Its localization is determined by the transmembrane domain (TMD) and tail domain at the C-terminus. We fused the TMD and the tail domain of Cb5-B to the C-terminus of a red fluorescent protein, tdTomato (tdTomato-CTT). When tdTomato-CTT was constitutively expressed under the ubiquitin10 promoter in Arabidopsis, the fluorescent signal was exclusively detected at the ER by means of the reliable ER marker SP-GFP-HDEL. Therefore, tdTomato-CTT can accurately visualize the ER in stable Arabidopsis lines. Additionally, transient assays showed that tdTomato-CTT can also be used as an ER marker in onion, rice, and Nicotiana benthamiana. We believe that TA proteins could be used to generate various organellar membrane markers in plants.

Structure and Mechanism of a Unique Diiron Center in Mammalian Stearoyl-CoA Desaturase.

Stearoyl-CoA desaturase 1 (SCD1) is a membrane-embedded metalloenzyme that catalyzes the formation of a double bond on a saturated acyl-CoA. SCD1 has a diiron center and its proper function requires an electron transport chain composed of NADH (or NADPH), cytochrome b5 reductase (b5R), and cytochrome b5 (cyt b5). Since SCD1 is a key regulator in fat metabolism and is required for survival of cancer cells, there is intense interest in targeting SCD1 for various metabolic diseases and cancers. Crystal structures of human and mouse SCD1 were reported recently; however, both proteins have two zinc ions instead of two iron ions in the catalytic center, and as a result, the enzymes are inactive. Here we report a general approach for incorporating iron into heterologously expressed proteins in HEK293 cells. We produced mouse SCD1 that contains a diiron center and visualized its diiron center by solving its crystal structure to 3.5 Å. We assembled the entire electron transport chain using the purified soluble domains of cyt b5 and b5R, and the purified mouse SCD1, and we showed that three proteins coordinate to produce proper products. These results established an in vitro system that allows precise perturbations of the electron transport chain for the understanding of the catalytic mechanism in SCD1.

Downregulation of CYB5D2 is associated with breast cancer progression.

We report here that CYB5D2 is associated with tumor suppression function in breast cancer (BC). CYB5D2 expression was significantly reduced in tamoxifen resistant MCF7 cells and in MCF7 cell-derived xenografts treated with TAM. CYB5D2 overexpression induced apoptosis in MCF7 cells; CYB5D2 knockdown enhanced MCF7 cell proliferation. Using the TCGA and Curtis datasets within the Oncomine database, CYB5D2 mRNA expression was downregulated in primary BCs vs breast tissues and HER2-positive or triple negative BCs vs estrogen receptor (ER)-positive BCs. Using the TCGA and Metabric datasets (n = 817 and n = 2509) within cBioPortal, 660 and 4891 differentially expressed genes (DEGs) in relation to CYB5D2 were identified. These DEGs were enriched in pathways governing cell cycle progression, progesterone-derived oocyte maturation, oocyte-meiosis, estrogen-mediated S-phase entry, and DNA metabolism. CYB5D2 downregulation decreased overall survival (OS, p = 0.0408). A CYB5D2-derived 21-gene signature was constructed and robustly correlated with OS shortening (p = 5.72e-12), and independently predicted BC deaths (HR = 1.28; 95% CI 1.08-1.52; p = 0.004) once adjusting for known clinical factors. CYB5D2 reductions displayed relationship with mutations in PIK3CA, GATA3, MAP3K1, CDH1, TP53 and RB1. Impressively, 85% (560/659) of TP53 mutations occurred in the 21-gene signature-positive BC. Collectively, we provide the first evidence that CYB5D2 is a candidate tumor suppressor of BC.

Molybdenum.

Molybdenum, a trace element essential for micro-organisms, plants, and animals, was discovered in 1778 by a Swedish chemist named Karl Scheele. Initially mistaken for lead, molybdenum was named after the Greek work molybdos, meaning lead-like. In the 1930s, it was recognized that ingestion of forage with high amounts of molybdenum by cattle caused a debilitating condition. In the 1950s, the essentiality of molybdenum was established with the discovery of the first molybdenum-containing enzymes. In humans, only 4 enzymes requiring molybdenum have been identified to date: sulfite oxidase, xanthine oxidase, aldehyde oxidase, and mitochondrial amidoxime-reducing component (mARC). Sulfite oxidase, an enzyme found in mitochondria, catalyzes oxidation of sulfite to sulfate, the final step in oxidation of sulfur amino acids (cysteine and methionine). Xanthine oxidase converts hypoxanthine to xanthine, and further converts xanthine to uric acid, preventing hypoxanthine, formed from spontaneous deamination of adenine, from leading to DNA mutations if paired with cytosine in place of thymine. Aldehyde oxidase is abundant in the liver and is an important enzyme in phase 1 drug metabolism. Finally, mARC, discovered less than a decade ago, works in concert with cytochrome b5 type B and NAD(H) cytochrome b5 reductase to reduce a variety of N-hydroxylated substrates, although the physiologic significance is still unclear. In the case of each of the molybdenum enzymes, activity is catalyzed via a tricyclic cofactor composed of a pterin, a dithiolene, and a pyran ring, called molybdenum cofactor (MoCo) (1).

Use of Phenoxyaniline Analogues To Generate Biochemical Insights into the Interactio n of Polybrominated Diphenyl Ether with CYP2B Enzymes.

Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.

Efficiency of the sulfate pathway in comparison to the Δ4- and Δ5-pathway of steroidogenesis in the porcine testis.

Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis.

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli.

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b5 at the N-terminal of interferon (b5-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b5 at the C-terminal of interferon (Met-IFN-b5-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b5-chimera), resolved this problem and gave enhanced biological activity.

Effect of lupeol on antioxidants and xenobiotic enzymes in N-Butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in experimental rats.

OBJECTIVE: Urothelial carcinoma of the bladder is a common malignancy ranked 9th with an estimated 356,600 new cases diagnosed annually worldwide. The study showed the protective effects of Lupeol in N-Butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in in vivo experimental model. Forty male healthy wistar rats were selected randomly divided into four groups. Group I rats served as healthy control. Group II rats were treated with BBN (150 mg/gavage/twice a week) for 8 weeks. Group III rats were treated with BBN + Lupeol [ Lupeol (50 mg/kg bw/day) treatment was started 1 week prior to the BBN treatment, and it was orally administered for 8 weeks]. Group IV rats were treated with Lupeol alone (50 mg/kg bw/day) for 8 weeks. All the experimental rats were maintained and euthanized at 32nd week. Serum and bladder tissues were collected and examined for biochemical parameters, serum markers and histopathological evaluation. Preventive (BBN + Lupeol) group modulates the activity of antioxidant enzymes such as Superoxide dismutase, Catalase, Reduced glutathione, Glutathione Peroxidase, Thiobarbituric acid reactive substances (TBARS) and drug metabolizing enzymes such as Cytochrome P450, Cytochrome b5, NADPH Cytochrome c reductase, NADPH- Quinone Oxidoreductase 1 and Glutathione-S-transferase when compared to BBN treated rats. Serological markers such as Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) were significantly (P&#60;0.05) decreased in preventive lupeol treated groups. Lupeol supplementation protects BBN induced bladder carcinogenesis in experimental rats by its antioxidant, anti-inflammatory and antiproliferative properties.

Structural and functional effects of cytochrome b5 interactions with human cytochrome P450 enzymes.

The small heme-containing protein cytochrome b5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein, solution NMR was used to identify b5 residues interacting with different human drug-metabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b5 α4-5 (CYP2A6 and CYP2E1) or this region and α2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b5 Mutations of key b5 residues suggest not only that different b5 surfaces are responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.

The cytochrome b5 CybE is regulated by iron availability and is crucial for azole resistance in A. fumigatus.

Cytochrome P450 enzymes (P450) play essential roles in redox metabolism in all domains of life including detoxification reactions and sterol biosynthesis. The activity of P450s is fuelled by two electron-transferring mechanisms, heme-independent P450 reductase (CPR) and the heme-dependent cytochrome b5 (CYB5)/cytochrome b5 reductase (CB5R) system. In this study, we characterised the role and regulation of the cytochrome b5 CybE in the fungal pathogen Aspergillus fumigatus. Deletion of the CybE encoding gene (cybE) caused a severe growth defect in two different A. fumigatus isolates, emphasising the importance of the CB5R system in this pathogen, while the non-essentiality of cybE indicates the partial redundancy of the CPR and CB5R systems. Interestingly, the growth defect caused by the cybE loss of function was even more drastic in A. fumigatus strain AfS77 compared to strain A1160P+ indicating a strain-dependent degree of compensation, which is supported by azole resistance studies. In agreement with CybE being important for the assistance of the ergosterol biosynthetic P450 Cyp51, deletion of cybE decreased resistance to the Cyp51-targeting antifungal voriconazole and caused accumulation of the ergosterol pathway intermediate eburicol. Northern analysis indicated that CybE deficiency leads to the compensatory transcriptional upregulation of Cyp51-encoding cyp51A and CPR-encoding cprA. Overexpression of cybE did not affect azole resistance suggesting that CybE activity is not rate limiting. Expression of cybE was found to be repressed during iron starvation by the iron-regulatory transcription factor HapX demonstrating iron dependence of CybE not only at the level of enzyme activity but also at the level of gene expression.

Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1.

Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome b5 (b5), but the exact role of b5 in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. b5 did not enhance reaction rates by decreasing the koff rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse-chase experiments. (S)-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC50 values for (S)-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, i.e. some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography.

Using Photoconvertible and Extractable Fluorescent Proteins to Study Autophagy in Plants.

Several methodologies have been employed to understand the kinetics of induced autophagic degradation in plants, but most of them are not capable of distinguishing the autophagic cargo proteins before and after induction of autophagy in cells. Here, we designed a mass photoconverter that allowed us to simultaneously monitor protein synthesis and degradation in tobacco BY-2 cells using a photoconvertible fluorescence marker protein, Kikume Green Red (KikGR). An example of a new protocol for the analysis of autophagy progression using a fusion protein of cytochrome b5 and KikGR under phosphate starvation is described. The other example described is the analysis of the proliferation of Golgi apparatus in tobacco BY-2 cells using the fusion protein of a prolyl 4-hydroxylase NtP4H1.1 and monomeric KikGR. A detailed protocol on key analysis, as well as tips and notes for experiments using KikGR proteins, are described.

2,3,7,8 Tetrachlorodibenzo-p-dioxin-induced RNA abundance changes identify Ackr3, Col18a1, Cyb5a and Glud1 as candidate mediators of toxicity.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is an aromatic, long-lived environmental contaminant. While the pathogenesis of TCDD-induced toxicity is poorly understood, it has been shown that the aryl hydrocarbon receptor (AHR) is required. However, the specific transcriptomic changes that lead to toxic outcomes have not yet been identified. We previously identified a panel of 33 genes that respond to TCDD treatment in two TCDD-sensitive rodent species. To identify genes involved in the onset of hepatic toxicity, we explored 25 of these in-depth using liver from two rat strains: the TCDD-resistant Han/Wistar (H/W) and the TCDD-sensitive Long-Evans (L-E). Time course and dose-response analyses of mRNA abundance following TCDD insult indicate that eight genes are similarly regulated in livers of both strains of rat, suggesting that they are not central to the severe L-E-specific TCDD-induced toxicities. The remaining 17 genes exhibited various divergent mRNA abundances between L-E and H/W strains after TCDD treatment. Several genes displayed a biphasic response where the initial response to TCDD treatment was followed by a secondary response, usually of larger magnitude in L-E liver. This secondary response was most often an exaggeration of the original TCDD-induced response. Only cytochrome b5 type A (microsomal) (Cyb5a) had equivalent TCDD sensitivity to the prototypic AHR-responsive cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1), while six genes were less sensitive. Four genes showed an early inter-strain difference that was sustained throughout most of the time course (atypical chemokine receptor 3 (Ackr3), collagen, type XVIII, alpha 1 (Col18a1), Cyb5a and glutamate dehydrogenase 1 (Glud1)), and of those genes examined in this study, are most likely to represent genes involved in the pathogenesis of TCDD-induced hepatotoxicity in L-E rats.