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Huntington's disease is characterized by a complex and heterogeneous pathogenic profile. Studies have shown that disturbance in lipid homeostasis may represent a critical determinant in the progression of several neurodegenerative disorders. The recognition of perturbed lipid metabolism is only recently becoming evident in HD. In order to provide more insight into the nature of such a perturbation and into the effect its modulation may have in HD pathology, we investigated the metabolism of Sphingosine-1-phosphate (S1P), one of the most important bioactive lipids, in both animal models and patient samples. Here, we demonstrated that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, we revealed that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro-survival actions seen after modulation of S1P-metabolizing enzymes allows this axis to emerge as a new druggable target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.
The existence of CYP5, CYP8A, and the CYP74 enzymes specialized for reaction with fatty acid peroxide substrates presents opportunities for a "different look" at the catalytic cycle of the cytochrome P450s. This review considers how the properties of the peroxide-metabolizing enzymes are distinctive, and how they tie in with those of the conventional monooxygenase enzymes. Some unusual reactions of each class have parallels in the other. As enzyme reactions and P450 structures emerge there will be possibilities for finding their special properties and edging this knowledge into the big picture.
A novel member of the plant cytochrome P450 CYP74 family of fatty acid hydroperoxide metabolizing enzymes has been cloned from melon fruit (Cucumis melo). The cDNA is comprised of 1,446 nucleotides encoding a protein of 481 amino acids. The homology at the amino acid level to other members of the CYP74 family is 35-50%, the closest relatives being allene oxide synthases. The cDNA was expressed in Escherichia coli, and the corresponding protein was purified by affinity column chromatography. The native enzyme showed a main Soret band at 418 nm, indicative of a low spin ferric cytochrome P450, and a 447-nm peak appeared in the CO-difference spectrum. Using [U-14C]radiolabeled substrate, HPLC, UV, and GC-MS, the products of conversion of 9S-hydroperoxy-linoleic acid were identified as 9-oxo-nonanic acid and 3Z-nonenal. Kinetic analysis of this hydroperoxide lyase showed the highest rate of reaction with 9-hydroperoxy-linolenic acid followed by 9-hydroperoxy-linoleic acid and then the corresponding 13-hydroperoxides. Overall, the newly characterized cytochrome P450 enzyme is a fatty acid hydroperoxide lyase with a preference, but not absolute specificity for the 9-positional hydroperoxides of linoleic and linolenic acids.
The mechanism of formation of 4-hydroxy-2E-nonenal (4-HNE) has been a matter of debate since it was discovered as a major cytotoxic product of lipid peroxidation in 1980. Recent evidence points to 4-hydroperoxy-2E-nonenal (4-HPNE) as the immediate precursor of 4-HNE (Lee, S. H., and Blair, I. A. (2000) Chem. Res. Toxicol. 13, 698-702; Noordermeer, M. A., Feussner, I., Kolbe, A., Veldink, G. A., and Vliegenthart, J. F. G. (2000) Biochem. Biophys. Res. Commun. 277, 112-116), and a pathway via 9-hydroperoxylinoleic acid and 3Z-nonenal is recognized in plant extracts. Using the 9- and 13-hydroperoxides of linoleic acid as starting material, we find that two distinct mechanisms lead to the formation of 4-H(P)NE and the corresponding 4-hydro(pero)xyalkenal that retains the original carboxyl group (9-hydroperoxy-12-oxo-10E-dodecenoic acid). Chiral analysis revealed that 4-HPNE formed from 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) retains >90% S configuration, whereas it is nearly racemic from 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9S-HPODE). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid is >90% S when derived from 9S-HPODE and almost racemic from 13S-HPODE. Through analysis of intermediates and products, we provide evidence that (i) allylic hydrogen abstraction at C-8 of 13S-HPODE leads to a 10,13-dihydroperoxide that undergoes cleavage between C-9 and C-10 to give 4S-HPNE, whereas direct Hock cleavage of the 13S-HPODE gives 12-oxo-9Z-dodecenoic acid, which oxygenates to racemic 9-hydroperoxy-12-oxo-10E-dodecenoic acid; by contrast, (ii) 9S-HPODE cleaves directly to 3Z-nonenal as a precursor of racemic 4-HPNE, whereas allylic hydrogen abstraction at C-14 and oxygenation to a 9,12-dihydroperoxide leads to chiral 9S-hydroperoxy-12-oxo-10E-dodecenoic acid. Our results distinguish two major pathways to the formation of 4-HNE that should apply also to other fatty acid hydroperoxides. Slight ( approximately 10%) differences in the observed chiralities from those predicted in the above mechanisms suggest the existence of additional routes to the 4-hydroxyalkenals.
Guava fruit was identified as a particularly rich source of 13-hydroperoxide lyase activity. The enzyme proved stable to chromatographic procedures and was purified to homogeneity. Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD. Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3' and 5' rapid amplification of cDNA ends. The sequence shows approximately 60-70% identity to known 13-hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5. The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl-beta-D-thiogalactopyranoside and delta-aminolevulinic acid. The expressed enzyme metabolized 13(S)-hydroperoxylinolenic acid over 10-fold faster than 13(S)-hydroperoxylinoleic acid and the 9-hydroperoxides of linoleic and linolenic acids. 13(S)-Hydroperoxylinolenic acid was converted to 12-oxododec-9(Z)-enoic acid and 3(Z)-hexenal, as identified by gas chromatography-mass spectrometry. The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was approximately 2000/s, comparable to values reported for the related allene oxide synthases. Distinctive features of the guava 13-hydroperoxide lyase and related cytochrome P450 are discussed.
Steroidogenic enzymes are differentially expressed throughout the ovarian cycle. The complex pattern of cell-specific up- and down-regulation accounts, at least in part, for the cyclic production of estrogens, androgens and progesterone. The gonadotropins follicle-stimulating hormone and luteinizing hormone are the main regulators of ovarian steroid hormone production and act primarily via the cAMP second-messenger system. Previous studies have identified cAMP-responsive sequences (CRS) in a number of genes encoding steroidogenic enzymes. In the present study we attempted to compare the cAMP responsiveness of some of these sequences with each other and with the classical cAMP-response element (CRE), as identified in the somatostatin gene. In addition, we were interested to determine whether or not the information for tissue-specific expression is contained by these sequences. Using transient transfection of reporter gene constructs, comprising the CRS of bCYP11A, bCYP17, hCYP21B and bovine adrenodoxin, we investigated cAMP-dependent and tissue-specific expression in primary cultures of bovine luteal and granulosa cells. Treatment of transfected luteal cells with forskolin markedly increased the expression of all but the CYP17-specific reporter gene constructs. A similar pattern of forskolin responsiveness was observed when these reporter gene constructs were transfected in bovine granulosa cells in primary culture. Furthermore, when a reporter gene construct containing the classical CRE genomic was transfected in bovine luteal cells, its expression was also highly stimulated upon treatment with forskolin. Thus, the classical cAMP/CRE system appears to be functional in these cells. Northern blot analysis of primary cultures of bovine luteal and granulosa cells revealed that bCYP17 and bCYP21B are not expressed in control and forskolin-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
The effect of dexamethasone on ACTH-induced accumulation of CYP11A and CYP17 mRNAs was studied in bovine adrenocortical cells in primary culture. The cells were treated with either ACTH (1 microM) or the adenylate cyclase activator forskolin (25 microM) and/or dexamethasone (100 nM). The accumulation of CYP11A and CYP17 mRNAs was evaluated by Northern blot analysis with the use of [alpha-32P]deoxy-CTP-labeled bovine CYP11A and CYP17 cDNAs. Chloramphenicol acetyltransferase (CAT) activity was monitored in bovine adrenocortical cells transfected with recombinant plasmids containing either CYP11A or CYP17 regulatory regions coupled to the CAT reporter gene and treated with forskolin and/or dexamethasone. Dexamethasone treatment of the cells cultured in the presence of ACTH or forskolin resulted in about 50% suppression of both CYP11A and CYP17 mRNA accumulation, with a concomitant fall in cortisol secretion to about 60% of the stimulated value. The effects of dexamethasone on accumulation of CYP11A and CYP17 mRNAs and cortisol secretion were blocked by pretreatment of the cells with RU 486 (100 nM), while RU 486 had no effect on forskolin-induced accumulation of either mRNA or cortisol secretion. Dexamethasone also inhibited the forskolin-induced expression of the transfected CYP11A- or CYP17-CAT constructs in bovine adrenocortical cells. The inhibitory effect of dexamethasone was greatly reduced by cotreatment of the transfected cells with RU 486. It is concluded that dexamethasone inhibits the ACTH-induced accumulation of CYP11A and CYP17 mRNAs at a transcriptional level and that the effect of dexamethasone is mediated by the glucocorticoid receptor.
Two soluble flavoproteins, purified from Escherichia coli cytosol and identified as flavodoxin and NADPH-flavodoxin (ferredoxin) reductase (flavodoxin reductase), have been found in combination to support the 17 alpha-hydroxylase activities of heterologously expressed bovine 17 alpha-hydroxylase cytochrome P450 (P450c17). Physical characteristics of the two flavoproteins including absorbance spectra, molecular weights, and amino-terminal sequences are identical with those reported previously for E. coli flavodoxin and flavodoxin reductase. Flavodoxin reductase, possessing FAD as a cofactor, is able to reconstitute P450c17 activities only in the presence of flavodoxin, an FMN-containing protein, and NAD(P)H. Reducing equivalents are utilized more effectively from NADPH than NADH by flavodoxin reductase. E. coli flavodoxin binds P450c17 directly and with relatively high affinity (apparent Ks approximately 0.2 microM) at low ionic strength, as evidenced by a change in spin state of the P450c17 heme iron upon titration with flavodoxin. This apparent spin shift is attenuated at moderate ionic strengths (100-200 mM KCl). In addition, bovine P450c17 binds reversibly to flavodoxin Sepharose in an ionic strength-dependent manner. These data implicate charge pairing as being important for the interaction between flavodoxin and P450c17. We propose that the amino acid sequence similarity between E. coli flavodoxin-flavodoxin reductase and the putative FMN, FAD, and NAD(P)H binding regions of cytochrome P450 reductase provides the basis for the reconstitution of P450c17 activities by this bacterial system.
Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17. It enhances the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17 alpha-hydroxyprogesterone to androstenedione by at least 10-fold. This latter activity is virtually undetectable in the absence of cytochrome b5. Other activities catalyzed by P450c17 include 17 alpha-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b5. The conversion of pregnenolone to 17 alpha-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17 alpha-hydroxyprogesterone is unchanged. These studies using purified systems suggest that cytochrome b5 plays a role in regulating the activities of P450c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis. In particular, they indicate that in human testes which contains a high b5/P450 ratio, 17 alpha-hydroxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b5 in the human testis, both the delta 4 and the delta 5 steroidogenic pathways can lead to testosterone production.
Steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha) mediates both 17 alpha-hydroxylase and 17,20-lyase activities. A relatively rare disease, 17 alpha-hydroxylase deficiency is characterized by defects in either or both of these activities. The molecular basis for variability of the defect is not well understood. We have determined the exonic sequence of the mutant P-450(17)alpha gene from one Japanese patient with combined 17 alpha-hydroxylase/17,20-lyase deficiencies. A stop codon (TGA) due to a single point mutation was found at the position of amino acid 17 in exon 1 of the P-450(17)alpha gene. The presence of a stop codon in the N-terminal region of this gene leads to the absence of a functional P-450(17)alpha protein in adrenal cortex and ovary, and consequently hypertension, primary amenorrhea and osteoporosis in this patient.