I We previously reported the gene deletion encoding cytochrome P-450 cholestcrol side-chain cleav... more I We previously reported the gene deletion encoding cytochrome P-450 cholestcrol side-chain cleavage enzyme (P-45OsCc, resulting in complete elimination of the adrenal gene expression and causing congenital adrenal hyperplasia in the rabbit. Using the rabbit congenital adrenal hyperplasia model, we investigated the wild type (wt) P-450,, gene dose effect on gene expression in three P-45OsCc genotype animals [wtht, wtlmutant (mt), mtlmt] identified by Southern blot analysis. Northern blots using a rabbit P-45OsCc cDNA probe revealed
During an investigation of plant products from the shrub Salsola tuberculata that affect adrenal ... more During an investigation of plant products from the shrub Salsola tuberculata that affect adrenal steroid hormone biosynthesis', an assay for the conversion of deoxycortiosterone to corticosterone by the adrenal 1 I/?-hydroxylase enzyme system (cytochrome P-45Oi is) was required for screening purposes. Since a large number of samples containing small amounts of highly labile compounds had to be screened, none of the assay procedures previously reported for this conversion2-7 was suitable. The main problems encountered with previously published methods were: time-consuming purification of relatively large amounts of enzymes required for multiple conversion assays, the separation of corticosterone, deoxycorticosterone, cofactors and inhibitors after incubation and accurate quantitation of steroids. The latter two problems were mainly caused by the interference of added plant extract. Several studies by other researchers2-5 showed that the enzymatic conversion of deoxycorticosterone to corticosterone by the adrenal mitochondrial 1 lfi-hydroxylase system requires the presence of oxygen, adrenodoxin reductase (ADXR), adrenodoxin (ADX), cytochrome P-45Or ia, NADPH ands an NADPH regenerating system such as glucose-6-phosphate, glucosed-phosphate dehydrogenase and magnesium chloride. The new method is based on the incubation of deoxycorticosterone with this reconstituted system in a total volume of 200 ,ul. After incubation steroids were separated and quantitated in one step using isocratic high-performance liquid chromatography (HPLC), Separation was achieved on a normal-phase silica column and detection of the steroids was done with a fixedwavelength UV detector at 254 nm. Recording and integration of chromatograms were performed with the aid of the data processing facility of the chromatograph. The use of HPLC allowed the rapid high-resolution separation of steroids in the incubation medium. Interference by test compounds with separation and quantitation, encountered with thin-layer chromatographic (TLC) separation and radiometric as well as fluorometric detection, was thus eliminated.
Baboon CYP11B1: The Localization and Catalytic Activity in Baboon Adrenal Tissue
Endocrine Research, 2002
A third gene encoding baboon CYP11B1 was isolated and was shown to catalyze only the metabolism o... more A third gene encoding baboon CYP11B1 was isolated and was shown to catalyze only the metabolism of deoxycorticosterone (DOC) to corticosterone. The investigation into the localization of CYP11B1 in the baboon adrenal tissue, using in situ hybridization, showed that mRNA transcripts were predominantly present in the zona reticularis (ZR) and zona fasciculata (ZF). Signal was also observed in the zona glomerulosa (ZG) and scattered within the medulla. Immunohistochemical studies, using rabbit anti-sheep CYP11B1 IgG, indicated that CYP11B1 was expressed only in the zona fasciculata, zona reticularis and in the medulla. CYP11B1 was not detected in the zona glomerulosa. Subsequent Western Blot investigations into the presence of CYP11B1 in baboon adrenal cortex and medullary homogenates indicated CYP11B1 as a single band in the cortex and as two distinct bands in the medulla. CYP11A was present only in the baboon adrenal cortex. The metabolism of deoxycorticosterone and corticosterone was subsequently investigated in the baboon adrenal cortex and medulla. In cortex homogenates, deoxycorticosterone was converted to corticosterone, and neither 18-hydroxycorticosterone nor aldosterone was detected. In medulla homogenates, however, corticosterone was metabolized to aldosterone, as confirmed by APcI-MS.
Allosteric interaction between 3β‐hydroxysteroid dehydrogenase/Δ 5 ‐Δ 4 isomerase and cytochrome b 5 influences cofactor binding
The FASEB Journal, Sep 14, 2012
The biosynthesis of steroid hormones, essential to the survival of all mammals, is dependent on t... more The biosynthesis of steroid hormones, essential to the survival of all mammals, is dependent on the activity of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD). 3βHSD activity is, in turn, influenced by cytochrome-b(5) (Cyt-b(5)). However, the mechanism through which this occurs is unknown. In this study, we investigated this mechanism by evaluating the influence of Cyt-b(5) on the dehydrogenase and isomerase activities of 3βHSD. Capra hircus 3βHSD was overexpressed in SF-9 cells, using a baculovirus expression system, and purified. Substrate and cofactor kinetics were determined spectrophotometrically in the presence and absence of purified Ovis aries liver Cyt-b(5). Nonspecific enzyme activity was evaluated by zero-enzyme, -substrate, and -cofactor blanks. Fusion proteins, 3βHSD-eCFP, and Cyt-b(5)-eYFP were subsequently coexpressed in COS-1 cells and analyzed for FRET. A CFP-YFP fusion protein served as positive control, while coexpression of 3βHSD-eCFP and cytochrome P450 17α-hydroxylase/17,20 lyase-eYFP (CYP17A1-eYFP) served as negative control. Results showed Cyt-b(5) to decrease the K(m,)(NAD(+)) value of 3βHSD ≈3.5-fold while increasing the V(max,app) of the dehydrogenase reaction ≈17%. FRET analysis showed COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b(5)-eYFP to exhibit a FRET signal ≈9-fold greater than that of the negative control. These results indicate that Cyt-b(5) augments 3βHSD activity via an allosteric mechanism by increasing the affinity of the enzyme toward NAD(+).
Sequence of the 11β-hydroxylase gene from the cape baboon (Papio Ursinus)
Endocrine Research, Nov 1, 1996
We investigated the nucleotide sequence of the steroid 11β-hydroxylase gene (CYP11B1) from the Ca... more We investigated the nucleotide sequence of the steroid 11β-hydroxylase gene (CYP11B1) from the Cape baboon (Papio ursinus). Six primers, previously used in studies on human CYP11B1, were utilised to amplify three overlapping fragments (A, B and C) of the baboon CYP11B1 by the polymerase chain reaction (PCR). Sequence analysis of the three fragments yielded the sequence of all the exons of baboon CYP11B1. The open reading frame of 1509 bases shows 57 nucleotide exchanges when compared to the human resulting in 18 amino acid substitutions. For eight of these exchanges we found the amino acid which is common for human aldosterone synthase at the corresponding position. Most of the remaining 10 amino acid substitutions were conservative. Eight of the substitutions were located in the first four exons with a cluster in the second half of exon 3. One substitution was in exon 5 (F280L) and the 10th was C494F at the end of the protein.
The rapid release of cortisol from the adrenal cortex upon ACTH receptor activation plays an inte... more The rapid release of cortisol from the adrenal cortex upon ACTH receptor activation plays an integral role in the stress response. It has been suggested that the quantitative control over adrenal steroidogenesis (quantity of total steroids produced) depends on the activities of cytochrome P450 side-chain cleavage and steroidogenic acute regulatory protein that supplies pregnenolone precursor to the pathway. The qualitative control (which steroids) then depends on the downstream steroidogenic enzymes, including cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17). In this review we focus on the relative contribution of P450c17 in the qualitative control of cortisol production with data collected from studies on South African Angora and Boer goats, as well as Merino sheep. Unique P450c17 genotypes were identified in these breeds with isoforms differing only with a couple of single amino acid residue substitutions. This review demonstrates how molecular and cellular differences relating to P450c17 activity can affect physiological and behavioural responses.
Cytochrome b5 forms homomeric complexes in living cells
The Journal of Steroid Biochemistry and Molecular Biology, Nov 1, 2012
Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome... more Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1). In the steroidogenic pathway CYP17A1 catalyses the metabolism of pregnenolone, yielding both glucocorticoid and androgen precursors. While not affecting the 17α-hydroxylation of pregnenolone, cyt-b(5) augments the 17,20 lyase reaction of 17-hydroxypregnenolone, catalyzing the formation of DHEA, through direct protein-protein interactions. In this study, multimeric complex formation of cyt-b(5) and the possible regulatory role of these complexes were investigated. Cyt-b(5) was isolated from ovine liver and used to raise anti-sheep cyt-b(5) immunoglobulins. Immunochemical studies revealed that, in vivo, cyt-b(5) is primarily found in the tetrameric form. Subsequent fluorescent resonance energy transfer (FRET) studies in COS-1 cells confirmed the formation of homomeric complexes by cyt-b(5) in live cells. Site-directed mutagenesis revealed that the C-terminal linker domain of cyt-b(5) is vital for complex formation. The 17,20-lyase activity of CYP17 was augmented by truncated cyt-b(5), which is unable to form complexes when co-expressed in COS-1 cells, thereby implicating the monomeric form of cyt-b(5) as the active species. This study has shown for the first time that cyt-b(5) forms homomeric complexes in vivo, implicating complex formation as a possible regulatory mechanism in steroidogenesis.
Scope: To determine the effect of Rooibos (Aspalathus linearis) on glucocorticoid biosynthesis an... more Scope: To determine the effect of Rooibos (Aspalathus linearis) on glucocorticoid biosynthesis and inactivation in vivo and in vitro. Methods and results: Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analyses of in vivo studies showed that human Rooibos consumption increased cortisone plasma levels in males (p = 0.0465) and reduced cortisol:cortisone ratios in males and females (p = 0.0486) at risk for cardiovascular disease. In rats, corticosterone (CORT) (p = 0.0275) and deoxycorticosterone (p = 0.0298) levels as well as the CORT:testosterone ratio (p = 0.0009) decreased following Rooibos consumption. The inactivation of cortisol was investigated in vitro by expressing 11-hydroxysteroid dehydrogenase type 1 (11 HSD1) and type 2 (11 HSD2) in CHO-K1 cells. Rooibos inhibited 11 HSD1, which resulted in a significant reduction in the cortisol:cortisone ratio (p < 0.01). No significant effect was detected on 11 HSD2. In vitro studies in adrenal H295R cells showed that Rooibos and rutin, one of the more stable flavonoid compounds present in Rooibos, significantly reduced the levels of cortisol and CORT in cells stimulated with forskolin to mimic a stress response. Conclusion: In vivo studies demonstrate that Rooibos significantly decreased glucocorticoid levels in rats and steroid metabolite ratios linked to metabolic disorders-cortisol:cortisone in humans and CORT:testosterone in rats. Results obtained at cellular level elucidate possible mechanisms by which these effects were achieved.
The Journal of Steroid Biochemistry and Molecular Biology, Apr 1, 2010
In adrenal steroidogenesis, CYP17 catalyses the 17␣-hydroxylation of pregnenolone and progesteron... more In adrenal steroidogenesis, CYP17 catalyses the 17␣-hydroxylation of pregnenolone and progesterone and the subsequent 17,20-lyase reaction, yielding adrenal androgens. The enzyme exhibits distinctly different selectivities towards these substrates in various species. CYP17 has also been shown to exhibit 16␣-hydroxylase activity towards progesterone in some species, with only human and chimp CYP17 catalysing the biosynthesis of substantial amounts of 16-OHprogesterone. The 16␣-hydroxylase activity was investigated by introducing an Ala105Leu substitution into human CYP17. The converse mutation, Leu105Ala was introduced into the baboon, goat and pig enzymes. Wt human CYP17 converted ∼30% progesterone to 16-OHprogesterone while the Ala105Leu mutant converted negligible amounts to 16-OHprogesterone (∼9%), comparable to wt CYP17 of the other three species when expressed in COS-1 cells. The ratio of 17-hydroxylated products to 16-OHprogesterone of human CYP17 was 2.7 and that of the mutant human construct 10.5. Similar ratios were observed for human and goat CYP17 with the corresponding Ala or Leu residues. Although the Leu105Ala mutation of both baboon and pig CYP17 exhibited the same trend regarding the ratios, the rate of progesterone conversion was reduced. Coexpression with cytochrome b 5 significantly decreased the ratio of 17-hydroxylated products to 16-OHprogesterone in the Leu105 constructs, while effects were negligible with Ala at this position. Homology models show that Ala105 faces towards the active pocket in the predicted B-C domain of CYP17. The smaller residue allows more flexibility of movement in the active pocket than Leu, presenting both the C16 and C17 of progesterone to the iron-oxy complex.
The Influence of the Amino Acid Substitution I98K on the Catalytic Activity of Baboon Cytochrome P450 Side‐Chain Cleavage (CYP11A1)
Endocrine Research, 2004
Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the first and &quot;rate-limiting&... more Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the first and &quot;rate-limiting&quot; step in steroidogenesis, the conversion of cholesterol to pregnenolone. In an effort to gain further insight into the structure/function relationship of this key enzyme, CYP11A1 was characterized in the Cape baboon (Papio ursinus), a species closely related to humans. Baboon cDNA was isolated from adrenal tissue and direct sequence analysis showed mature baboon and human CYP11A1 share 98% deduced amino acid homology. The cDNA was subsequently amplified and two recombinant constructs, CYP11A1a and CYP11A1b, were cloned. Sequence analyses of the constructs revealed four amino acid substitutions. The constructs were expressed in nonsteroidogenic mammalian COS-1 cells with 25-hydroxycholesterol as substrate. Apparent Km values of 1.62 and 4.53 microM were determined for CYP11A1a and CYP11A1b, respectively. Homology modeling revealed that the lower substrate affinity of CYP11B1b could be attributed to an I98K substitution, which lies between the B and C helices, providing further evidence for the importance of this domain in the catalytic activity of CYP11A1.
Hypocortisolism in the South African Angora goat: The role of 3βHSD
Molecular and Cellular Endocrinology, Feb 1, 2010
South African Angora goats are susceptible to cold stress, due to their inability to produce suff... more South African Angora goats are susceptible to cold stress, due to their inability to produce sufficient levels of cortisol. During adrenal steroidogenesis the production of cortisol relies on the activity of two key enzymes, namely cytochrome P450 17alpha-hydroxylase and 3beta-hydroxysteroid dehydrogenase. Cytochrome P450 17alpha-hydroxylase has previously been identified as a factor contributing to hypocortisolism in the South African Angora goat. In this comparative study, the catalytic activity of Angora and ovine 3beta-hydroxysteroid dehydrogenase, which differ by five amino acid residues, was characterized. The conversion of 17-hydroxypregnenolone and dehydroepiandosterone to their corresponding products, 17-hydroxyprogesterone and androstenedione, by the two enzymes differed significantly. The enzymes were subsequently co-expressed with Angora P450 17alpha-hydroxylase. Major differences were observed in pregnenolone metabolism with a significant reduction in the formation of the cortisol precursor, 17-hydroxyprogesterone, by cells expressing Angora 3beta-hydroxysteroid dehydrogenase, implicating 3beta-hydroxysteroid dehydrogenase as an additional factor contributing to hypocortisolism in the South African Angora goat.
Cytochrome b 5 (cyt-b 5) is essential for the regulation of steroidogenesis and as such has been ... more Cytochrome b 5 (cyt-b 5) is essential for the regulation of steroidogenesis and as such has been implicated in a number of clinical conditions. It is well documented that this small hemoprotein augments the 17,20-lyase activity of cytochrome P450 17αhydroxylase/17,20-lyase (CYP17A1). Studies have revealed that this augmentation is accomplished by cyt-b 5 enhancing the interaction between cytochrome P450 reductase (POR) and CYP17A1. In this paper we present evidence that cyt-b 5 induces a conformational change in CYP17A1, in addition to facilitating the interaction between CYP17A1 and POR. We also review the recently published finding that cyt-b 5 allosterically augments the activity of 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4 isomerase (3βHSD), a non cytochrome P450 enzyme, by increasing the enzymes affinity for its cofactor, NAD +. The physiological importance of this finding, in terms of understanding adrenal androstenedione production, is examined. Finally, evidence that cyt-b 5 is able to form homomeric complexes in living cells is presented and discussed.
Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the conversion of cholesterol to pregneno... more Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the conversion of cholesterol to pregnenolone, the first step in steroidogenesis. The absence of a solved crystal structure has complicated deductions pertaining to the structure/function relationships of this key enzyme. Although a number of techniques have been employed to identify domains and specific amino acid residues important for catalytic activity, these methods have been unsuccessful in predicting three-dimensional orientations in space and thus the mechanism by which they exert their kinetic effect. This review aims to demonstrate the significant contribution homology modelling, when employed as a tool in combination with other standard biochemical techniques, has made towards our understanding of CYP11A1.
Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critic... more Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5a-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown. We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5a-reduced C19 steroids-11OH-5a-androstanedione, 11keto-5a-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1 nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.
The metabolism of progesterone (PROG) by cytochrome P450 17␣-hydroxylase/17,20-lyase (CYP17A1) re... more The metabolism of progesterone (PROG) by cytochrome P450 17␣-hydroxylase/17,20-lyase (CYP17A1) results in the formation of both 17␣-hydroxyprogesterone (17-OHPROG) and 16␣-hydroxyprogesterone (16-OHPROG) in humans. Unlike 17-OHPROG, 16-OHPROG is not metabolised further in steroidogenic tissue. While this metabolite can be readily detected in serum and urine, its physiological role remains unclear. This paper reviews the production of 16-OHPROG by human CYP17A1 by providing insight into the catalysis of PROG by CYP17A1 and highlights the role of Ala105 in the 16␣-hydroxylation reaction. As 16-OHPROG has been putatively linked to reproductive function, we investigated the interaction of this steroid metabolite with both isoforms of the human progesterone receptor (hPR). We show for the first time that 16-OHPROG can bind to both hPR-A and hPR-B and act as an agonist for both receptors.
Biological activities of the shrubSalsola tuberculatiformis Botsch.: Contraceptive or stress alleviator?
BioEssays, May 19, 2003
Plants belonging to the genus Salsola (Family: Chenopodiaceae) are common in the arid and semiari... more Plants belonging to the genus Salsola (Family: Chenopodiaceae) are common in the arid and semiarid regions of our planet with no less than 69 different Salsola species found in Namibia and the Republic of South Africa. This genus is used as a traditional medicine and aqueous extracts of Salsola have been used by Bushmen women as an oral contraceptive. Ingestion of the Namibian shrub Salsola tuberculatiformis Botsch. by pregnant Karakul sheep leads to prolonged gestation and fetal post-maturity and, as a result, the pelts of the new-born karakul lambs are worthless. This initiated an investigation into the active agents in the plant, using the terminal enzyme in adrenal corticosteroidogenesis, cytochrome P450-dependent 11beta-hydroxylase (P450c11), as a bioassay. Although the active fraction, S2, was extremely labile, partial structure determination suggested the presence of synephrine and a highly reactive aziridine. Therefore a more stable analogue, 2-(4-acetoxyphenyl)2-chloro-N-methylethylammonium-chloride (compound A), was synthesised, which, like the active plant extracts, inhibited adrenal steroidogenesis and acted as a contraceptive. In addition, compound A was stabilised by interaction with steroid-binding globulins in plasma thus enhancing biological activity in vivo. These findings provided explanations for the complex biological effects of the shrub as well as a new insight into the mode of action of chemically labile plant products in vivo.
Functional expression and characterisation of human cytochrome P45017α in Pichia pastoris
Journal of Biotechnology, May 1, 2007
Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses... more Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17. Heterologous expression systems have contributed greatly to our current knowledge of CYP17&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s dual catalytic activity. However, due to the hydrophobic nature of this membrane-bound protein, primarily truncated and modified forms of CYP17 are currently being expressed heterologously. Although the N-terminally modified enzyme has been well characterised, protein structure and function studies still necessitate the expression of unmodified, wild-type CYP17. We report here the expression of a catalytically active, unmodified human CYP17 in the industrial methylotrophic yeast, Pichia pastoris. A typical P450 carbon monoxide difference spectrum, with an absorption maximum at 448nm and a substrate-induced type I spectrum were recorded using a detergent-solubilised cellular fraction containing CYP17. The expressed enzyme catalysed the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone, a product unique to human and chimpanzee CYP17. This is the first report showing the heterologous expression of a fully functional human steroidogenic cytochrome P450 enzyme in P. pastoris.
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Papers by Pieter Swart