Degradation of FATTY ACID EXPORT PROTEIN1 by RHOMBOID-LIKE PROTEASE11 contributes to cold tolerance in Arabidopsis.

Plants need to acclimate to different stresses to optimize growth under unfavorable conditions. In Arabidopsis (Arabidopsis thaliana), the abundance of the chloroplast envelope protein FATTY ACID EXPORT PROTEIN1 (FAX1) decreases after the onset of low temperatures. However, how FAX1 degradation occurs and whether altered FAX1 abundance contributes to cold tolerance in plants remains unclear. The rapid cold-induced increase in RHOMBOID-LIKE PROTEASE11 (RBL11) transcript levels, the physical interaction of RBL11 with FAX1, the specific FAX1 degradation after RBL11 expression, and the absence of cold-induced FAX1 degradation in rbl11 loss-of-function mutants suggest that this enzyme is responsible for FAX1 degradation. Proteomic analyses showed that rbl11 mutants have higher levels of FAX1 and other proteins involved in membrane lipid homeostasis, suggesting that RBL11 is a key element in the remodeling of membrane properties during cold conditions. Consequently, in the cold, rbl11 mutants show a shift in lipid biosynthesis toward the eukaryotic pathway, which coincides with impaired cold tolerance. To test whether cold sensitivity is due to increased FAX1 levels, we analyzed FAX1 overexpressors. The rbl11 mutants and FAX1 overexpressor lines show superimposable phenotypic defects upon exposure to cold temperatures. Our re-sults show that the cold-induced degradation of FAX1 by RBL11 is critical for Arabidop-sis to survive cold and freezing periods.

Fatty acid export (FAX) proteins contribute to oil production in the green microalga Chlamydomonas reinhardtii.

In algae and land plants, transport of fatty acids (FAs) from their site of synthesis in the plastid stroma to the endoplasmic reticulum (ER) for assembly into acyl lipids is crucial for cellular lipid homeostasis, including the biosynthesis of triacylglycerol (TAG) for energy storage. In the unicellular green alga Chlamydomonas reinhardtii, understanding and engineering of these processes is of particular interest for microalga-based biofuel and biomaterial production. Whereas in the model plant Arabidopsis thaliana, FAX (fatty acid export) proteins have been associated with a function in plastid FA-export and hence TAG synthesis in the ER, the knowledge on the function and subcellular localization of this protein family in Chlamydomonas is still scarce. Among the four FAX proteins encoded in the Chlamydomonas genome, we found Cr-FAX1 and Cr-FAX5 to be involved in TAG production by functioning in chloroplast and ER membranes, respectively. By in situ immunolocalization, we show that Cr-FAX1 inserts into the chloroplast envelope, while Cr-FAX5 is located in ER membranes. Severe reduction of Cr-FAX1 or Cr-FAX5 proteins by an artificial microRNA approach results in a strong decrease of the TAG content in the mutant strains. Further, overexpression of chloroplast Cr-FAX1, but not of ER-intrinsic Cr-FAX5, doubled the content of TAG in Chlamydomonas cells. We therefore propose that Cr-FAX1 in chloroplast envelopes and Cr-FAX5 in ER membranes represent a basic set of FAX proteins to ensure shuttling of FAs from chloroplasts to the ER and are crucial for oil production in Chlamydomonas.

Underestimated reactions and regulation patterns of adrenal cytochromes P450.

Although cytochrome P450 (CYP) systems including the adrenal ones are being investigated since many years, there are still reactions and regulation patterns that have been underestimated ever since. This review discusses neglected ones to bring them into the focus of investigators working in the field. Novel substrates and reactions described for adrenal CYPs recently point to the fact that different from what has been believed for many years, adrenal CYPs are less selective than previously thought. The conversion of steroid sulfates, intermediates of steroid biosynthesis as well as of exogenous compounds are being discussed here in more detail and consequences for further studies are drawn. Furthermore, it was shown that protein-protein interactions may have an important effect not only on the activity of adrenal CYPs, but also on the product pattern of the reactions. It was found that, as expected, the stoichiometry of CYP:redox partner plays an important role for tuning the activity. In addition, competition between different CYPs for the redox partner and for electrons and possible alterations by mutants in the efficiency of electron transfer play an important role for the activity and product pattern. Moreover, the influence of phosphorylation and small charged molecules like natural polyamines on the activity of adrenal systems has been demonstrated in-vitro indicating a possible regulation of adrenal CYP reactions by affecting redox partner recognition and binding affinity. Finally, an effect of the genetic background on the consequences of mutations in adrenal CYPs found in patients was suggested from corresponding in-vitro studies indicating that a different genetic background might be able to significantly affect the activity of a CYP mutant.

Plant membrane-protein mediated intracellular traffic of fatty acids and acyl lipids.

In plants, de novo synthesis of fatty acids (FAs) occurs in plastids, whereas assembly and modification of acyl lipids is accomplished in the endoplasmic reticulum (ER) and plastids as well as in mitochondria. Subsequently, lipophilic compounds are distributed within the cell and delivered to their destination site. Thus, constant acyl-exchanges between subcellular compartments exist. These can occur via several modes of transport and plant membrane-intrinsic proteins for FA/lipid transfer have been shown to play an essential role in delivery and distribution. Lately, substantial progress has been made in identification and characterization of transport proteins for lipid compounds in plant organelle membranes. In this review, we focus on our current understanding of protein mediated lipid traffic between organelles of land plants.

The impact of the clinical CYP11B2 mutation V386A strongly depends on the enzyme's genetic background.

Human cytochrome P450 11B2 (CYP11B2) is an essential enzyme in the steroid hormone biosynthesis, which catalyzes the last three reaction steps of the aldosterone synthesis. These reactions comprise a hydroxylation at position C11 of the steroid intermediate deoxycorticosterone yielding corticosterone, followed by a hydroxylation at position C18 yielding 18-hydroxy-corticosterone and a subsequent oxidation of the hydroxyl group at C18, which results in the formation of aldosterone. Alterations in the amino acid sequence of CYP11B2 often cause severe disease patterns. We previously described a procedure for expression and purification of human CYP11B2 employing recombinant E. coli, which allows the rapid characterization of CYP11B2 mutants on a molecular level. This system was now utilized for the examination of the influence of the polymorphism at position 173 in combination with the mutation V386A on the activity of CYP11B2. Our in vitro findings show that the combination of the V386A mutation with the variant CYP11B2 173Arg only slightly reduces the 18-hydroxylase and 18-oxidase activity, whereas the V386A mutation with the CYP11B2 173Lys variant almost abolishes the 18-hydroxylation and 18-oxidation. In both cases the 11-hydroxylase activity is not affected. These findings highlight the importance of the genetic background of an enzyme when regarding the effect of clinical mutations.

The role of sulfated steroid hormones in reproductive processes.

Sulfated steroid hormones, such as dehydroepiandrosterone sulfate or estrone-3-sulfate, have long been regarded as inactive metabolites as they cannot activate classical steroid receptors. Some of them are present in the blood circulation at quite high concentrations, but generally sulfated steroids exhibit low membrane permeation due to their hydrophilic properties. However, sulfated steroid hormones can actively be imported into specific target cells via uptake carriers, such as the sodium-dependent organic anion transporter SOAT, and, after hydrolysis by the steroid sulfatase (so-called sulfatase pathway), contribute to the overall regulation of steroid responsive organs. To investigate the biological significance of sulfated steroid hormones for reproductive processes in humans and animals, the research group "Sulfated Steroids in Reproduction" was established by the German Research Foundation DFG (FOR1369). Projects of this group deal with transport of sulfated steroids, sulfation of free steroids, desulfation by the steroid sulfatase, effects of sulfated steroids on steroid biosynthesis and membrane receptors as well as MS-based profiling of sulfated steroids in biological samples. This review and concept paper presents key findings from all these projects and provides a broad overview over the current research on sulfated steroid hormones in the field of reproduction.

Role of steroid sulfatase in steroid homeostasis and characterization of the sulfated steroid pathway: Evidence from steroid sulfatase deficiency.

The impact of steroid sulfatase (STS) activity in the circulating levels of both sulfated and unconjugated steroids is only partially known. In addition, the sulfated steroid pathway, a parallel pathway to the one for unconjugated steroids, which uses the same enzymes, has never been characterized in detail before. Patients with steroid sulfatase deficiency (STSD) are unable to enzymatically convert sulfated steroids into their unconjugated forms, and are a good model to elucidate how STS affects steroid biosynthesis and to study the metabolism of sulfated steroids. We quantified unconjugated and sulfated steroids in STSD serum, and compared these results with data obtained from serum of healthy controls. Most sulfated steroids were increased in STSD. However, androstenediol-3-sulfate and epiandrosterone sulfate showed similar levels in both groups, and the concentrations of androsterone sulfate were notably lower. Hydroxylated forms of DHEAS and of pregnenolone sulfate were found to be increased in STSD, suggesting a mechanism to improve the excretion of sulfated steroids. STSD testosterone concentrations were normal, but cholesterol and DHEA were significantly decreased. Additionally, serum bile acids were three-fold higher in STSD. Correlations between concentrations of steroids in each group indicate that 17α-hydroxy-pregnenolone-3-sulfate in men is mainly biosynthesized from the precursor pregnenolone sulfate and androstenediol-3-sulfate from DHEAS. These findings confirm the coexistence of two steroidogenic pathways: one for unconjugated steroids and another one for sulfated steroids. Each pathway is responsible for the synthesis of specific steroids. The equal levels of testosterone, and the reduced level of unconjugated precursors in STSD, support that testosterone is primarily synthesized from sulfated steroids. In consequence, testosterone synthesis in STSD relies on an enzyme with sulfatase activity other than STS. This study reveals that STS is a key player of steroid biosynthesis regulating the availability of circulating cholesterol.

A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

Metabolism of Oral Turinabol by Human Steroid Hormone-Synthesizing Cytochrome P450 Enzymes.

The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco- and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17ß-hydroxy-17α-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 µM and 5.4 µM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min(-1) mM(-1) for CYP11A1, 741 min(-1) mM(-1) for CYP11B1, and 3338 min(-1) mM(-1) for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli-based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11ß-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11ß,18-diOH-OT and 11ß-OH-OT-18-al, which rearranges to its tautomeric form 11ß,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography-tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies.

Dehydroepiandrosterone sulfate (DHEAS) stimulates the first step in the biosynthesis of steroid hormones.

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant circulating steroid in human, with the highest concentrations between age 20 and 30, but displaying a significant decrease with age. Many beneficial functions are ascribed to DHEAS. Nevertheless, long-term studies are very scarce concerning the intake of DHEAS over several years, and molecular investigations on DHEAS action are missing so far. In this study, the role of DHEAS on the first and rate-limiting step of steroid hormone biosynthesis was analyzed in a reconstituted in vitro system, consisting of purified CYP11A1, adrenodoxin and adrenodoxin reductase. DHEAS enhances the conversion of cholesterol by 26%. Detailed analyses of the mechanism of DHEAS action revealed increased binding affinity of cholesterol to CYP11A1 and enforced interaction with the electron transfer partner, adrenodoxin. Difference spectroscopy showed K(d)-values of 40 ± 2.7 µM and 24.8 ± 0.5 µM for CYP11A1 and cholesterol without and with addition of DHEAS, respectively. To determine the K(d)-value for CYP11A1 and adrenodoxin, surface plasmon resonance measurements were performed, demonstrating a K(d)-value of 3.0 ± 0.35 nM (with cholesterol) and of 2.4 ± 0.05 nM when cholesterol and DHEAS were added. Kinetic experiments showed a lower Km and a higher kcat value for CYP11A1 in the presence of DHEAS leading to an increase of the catalytic efficiency by 75%. These findings indicate that DHEAS affects steroid hormone biosynthesis on a molecular level resulting in an increased formation of pregnenolone.

Carboetomidate: an analog of etomidate that interacts weakly with 11ß-hydroxylase.

BACKGROUND: Carboetomidate is a pyrrole etomidate analog that is 3 orders of magnitude less potent an inhibitor of in vitro cortisol synthesis than etomidate (an imidazole) and does not inhibit in vivo steroid production. Although carboetomidate's reduced functional effect on steroid synthesis is thought to reflect lower binding affinity to 11ß-hydroxylase, differential binding to this enzyme has never been experimentally demonstrated. In the current study, we tested the hypothesis that carboetomidate and etomidate bind with differential affinity to 11ß-hydroxylase by comparing their abilities to inhibit photoaffinity labeling of purified enzyme by a photoactivatable etomidate analog and to modify the enzyme's absorption spectrum in a way that is indicative of ligand binding. In addition, we made a preliminary exploration of the manner in which etomidate and carboetomidate might differentially interact with this site using spectroscopic methods as well as molecular modeling techniques to better understand the structural basis for their selectivity. METHODS: The ability of azi-etomidate to inhibit cortisol synthesis was tested by assessing its ability to inhibit cortisol synthesis by H295R cells. The binding affinities of etomidate and carboetomidate to 11ß-hydroxylase were compared by assessing their abilities to (1) inhibit photoincorporation of the photolabile etomidate analog [(3)H]azi-etomidate into the enzyme and (2) modify the absorption spectrum of the enzyme's heme group. In silico docking studies of etomidate, carboetomidate, and azi-etomidate binding to 11ß-hydroxylase were performed using the computer software GOLD. RESULTS: Similar to etomidate, azi-etomidate potently inhibits in vitro cortisol synthesis. Etomidate inhibited [(3)H]azi-etomidate photolabeling of 11ß-hydroxylase in a concentration-dependent manner. At a concentration of 40 µM, etomidate reduced photoincorporation of [(3)H]azi-etomidate by 96% ± 1% whereas carboetomidate had no experimentally detectable effect. On addition of etomidate to 11ß-hydroxylase, a type 2 difference spectrum was produced indicative of etomidate complexation with the enzyme's heme iron; carboetomidate had no effect whereas azi-etomidate produced a reverse type 1 spectrum. Computer modeling studies predicted that etomidate, carboetomidate, and azi-etomidate can fit into the heme-containing pocket that forms 11ß-hydroxylase's active site and pose with their carbonyl oxygens interacting with the heme iron and their phenyl rings stacking with phenylalanine-80. However, additional unique poses were identified for etomidate and azi-etomidate that likely account for their higher affinities. CONCLUSIONS: Carboetomidate's reduced ability to suppress in vitro and in vivo steroid synthesis as compared with etomidate reflects its lower binding affinity to 11ß-hydroxylase and may be attributed to carboetomidate's inability to form a coordination bond with the heme iron located at the enzyme's active site.