Maliyakal E. John

Regulation of Synthesis and Activity of Cytochrome P-450 Enzymes in Physiological Pathways

Regulation of the levels and activities of various forms of cytochrome P450 has important implications in human biology. For example, it is well known that different individuals have different capacities to metabolize various drugs.1 Such variation may result, in part, from an allelic distribution of P-450 isozymes;2 or it may also result from individual variations in the level of a specific isozyme.3,4 Also, deficiencies of different steroid hydroxylases lead to various disease states. For example, adrenal hyperplasia occurs in approximately one in 5000 births, making it one of the more common inborn errors of metabolism.5 In at least 95% of these cases, the deficiency is detected as a decrease in steroid 21-hydroxylation in the adrenal cortex,6 a reaction catalyzed by a specific form of P-450 (P-450C21), leading to deficient cortisol biosynthesis. These are but a few examples which illustrate the reasons why investigation of the regulation of P-450 activities has become such an active area of research. In each example, understanding of the molecular basis of variations in P-450 gene expression will be necessary for the complete elucidation of the different phenotypes.

Structural characterization of the bovine CYP17 (17α-hydroxylase) gene

Abstract The complete exonic and partial intronic sequence of the bovine CYP17 (P450 17α ) gene has been determined. The gene contains eight exons with exon/intron boundaries which are identical to those determined previously for the human CYP17 gene. The site of initiation of transcription of this gene is located within a 6-base sequence 52 bp from the initiation of translation. Considerable sequence homology (58.7%) is found when ~500 bp of the 5′-flanking sequences of the bovine and human CYP17 genes are compared. A computer-based search of this region of bovine CYP17 for consensus sequences associated with binding of transcription factors (i.e., GR, PR, CREB/ATF, AP1, AP2, AP3, AP4, AP5, OTF, CTF/NF1, SP1) shows only the consensus CREB/ATF sequence TGACGT which is also found to be at approximately the same position in the human CYP17 gene. In bovine adrenal cortex, transcription of the CYP17 gene is regulated by the peptide hormone adrenocorticotropin via cAMP. Whether the consensus CREB/ATF sequence is associated with the cAMP-mediated transcription of the CYP17 gene remains to be elucidated.

Ontogeny of adrenal steroid hydroxylases: evidence for cAMP-independent gene expression

Total RNA from normal and anencephalic human fetal adrenals was examined by blot analysis for transcripts encoding P-450scc, P-450(11) beta, P-450(17) alpha, P-450C21 and adrenodoxin using bovine cDNA clones specific for these different enzymes. The specific contents of RNA encoding these components of the adrenocortical steroidogenic pathway were found to be similar in both types of adrenal tissue. Likewise, immunoblot analysis showed comparable concentrations of P-450scc, P450(17) alpha and adrenodoxin protein to be present in adrenal tissues from normal and anencephalic human fetuses. Immunoblot analysis of homogenates of fetal sheep adrenals of increasing gestational age (85-145 days) showed constant levels of P-450scc and P-450(11) beta, but increasing P-450(17) alpha content, especially near term. Both sheep fetuses prior to 136 days gestational age and human anencephalic fetuses are known to have extremely low circulating levels of immunoreactive ACTH as well as very low adrenal adenylate cyclase activity. Thus, it is concluded that factors other than pituitary ACTH which operate independent of adenylate cyclase activation are required for the initial expression (imprinting) of steroid hydroxylase genes.

Regulation of the biosynthesis of steroidogenic enzymes.

Recombinant DNA technology can permit study of the regulation of steroid hydroxylase gene expression at three levels. The first of these is cAMP-regulated gene expression. In the adrenal, ACTH, via cAMP, increases the expression of the genes for all of the cytochrome P-450 species involved in the steroid biosynthetic pathway, as well as the iron-sulfur protein, adrenodoxin. This action of cAMP is inhibited by cycloheximide, suggestive of the involvement of a regulatory protein factor in mediating this action of cAMP. The second level is tissue-specific regulation of steroid hydroxylase gene expression. An example of this which we have studied is the expression of cholesterol side-chain cleavage cytochrome P-450 (P-450sec) and 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) in the bovine ovary. P-450sec is expressed at high levels in the corpus luteum but at low levels in follicles, whereas P-450(17)alpha is expressed in follicles, but is undetectable in the corpus luteum. The third level is fetal imprinting. A number of the cytochrome P-450 species involving in the steroidogenic pathway are expressed in the fetal adrenal at a time when exposure of the gland to ACTH is very low, suggestive that factor(s) other than pituitary ACTH mediate this expression in fetal life.

Regulation of cholesterol side-chain cleavage cytochrome P-450 gene expression in adrenal cells in monolayer culture

Utilizing a cDNA probe specific for bovine cytochrome P-450scc and primary monolayer cultures of bovine adrenocortical cells and human fetal adrenal cells, it has been shown that the chronic action of ACTH on the adrenal cortex includes regulation of P-450scc gene expression at the transcriptional level. The bovine P-450scc cDNA hybridizes strongly to human, pig and rat RNA. Advantage was taken of the cross-reactivity of the bovine P-450scc cDNA with human P-450scc RNA to examine the regulation of P-450scc gene expression by ACTH in human fetal adrenal cells. This process is mediated by cyclic AMP and is inhibited by cycloheximide, in a fashion similar to bovine adrenocortical cells, suggestive that a protein factor(s) activates the response in both species. Hence, the actions of ACTH to regulate P-450scc gene expression in bovine adult adrenocortical cells and human fetal adrenal cells appear to proceed by similar mechanisms.

Effect of ACTH on the stability of mRNAs encoding bovine adrenocortical P-450scc, P-45011β, P-45017α, P-450C21 and adrenodoxin

ACTH treatment of bovine adrenocortical cells in primary culture causes increased accumulation of mRNAs encoding cytochromes P-450 scc , P-450 11 β , P-450 17 α , P-450 C21 and adrenodoxin as well as increased transcriptional activity of their respective genes. In this study we have shown that ACTH does not greatly affect the half-life of mRNAs encoding P-450 11 β , P-450 17 α , P-450 C21 and adrenodoxin. However, in the case of P-450 scc mRNA, ACTH causes a five-fold increase in the half-life leading to a significant stabilization of P450 scc mRNA. Thus it appears that the levels of mRNAs encoding P-450 11 β , P-450 17 α , P-450 C21 and adrenodoxin are regulated by ACTH primarily at the transcriptional level, while that for P-450 scc is regulated at both the transcriptional and post-transcriptional levels.

Oxidation-reduction properties of the hemoglobin of the opossum, Didelphis virginiana

Abstract 1. 1. Isolated opossum hemoglobin is found to be more susceptible to oxidation by several oxidizing agents than is human hemoglobin A. 2. 2. Opossum methemoglobin is found to be more susceptible to reduction by several reducing agents than is human hemoglobin A. 3. 3. These results are due to amino acid differences between the two hemoglobins, including the replacement of the distal histidine residue in the alpha chains of hemoglobin A by a glutamine residue in these subunits of opossum hemoglobin. 4. 4. Such differences also appear to be responsible for the ability of the opossum red cell NADH methemoglobin reductase to maintain opossum hemoglobin in the functional ferrous form.

Molecular Cloning of Steroid Hydroxylases

Recombinant plasmids specific for bovine adrenocortical cytochromes P-450scc and P-45011 beta have been identified and characterized. Using these cDNA inserts as probes, it is found that tissue specificity of gene expression for these two proteins is as expected. Cytochrome P-450scc mRNA is found in adrenocortical and corpus luteum RNA while cytochrome P-45011 beta mRNA is found only in adrenocortical RNA. Neither mRNA was detected in heart, liver or kidney RNA. Cytochrome P-450scc mRNA is found to be 1.9 kb in length while cytochrome P-45011 beta mRNA is found to be 4 kb in length. Treatment of bovine adrenocortical cells with ACTH or other modulators results in increased levels of cytochrome P-450scc and cytochrome P-45011 beta mRNAs. Cytochrome P-450scc is found to be encoded by a limited number or even a single gene.

Differential effects of pH and inositol hexaphosphate on the spectroscopic properties of the alpha and beta subunits in methemoglobins M Milwaukee and A.

The effect of pH and inositol hexaphosphate on the electron spin resonance spectra of the alpha-hemes (g = 6.0) and the beta-hemes (g = 6.7) has been measured in methemoglobin M Milwaukee and compared with that of methemoglobin A (g = 6.0). The beta-hemes are found to be comparatively insensitive to both effectors while the alpha-hemes behave in a manner similar to the heme groups of methemoglobin A. Binding of inositol hexaphosphate enhances the high spin ESR signal of the alpha-hemes in both methemoglobins. Comparison of the optical properties of methemoglobins A and M Milwaukee over the pH range from 5.0 to 8.1 shows that inositol hexaphosphate has a differential effect on the subunit types in these two methemoglobins. At low pH the spectral changes observed upon inositol hexaphosphate binding arise primarily from the beta-hemes, while at neutral and alkaline pH these changes arise from both subunit types. The beta-heme spectral changes appear to be pH independent while those arising from the alpha-hemes are strongly pH dependent. It is concluded that it is the hydroxymet form of the alpha-hemes which undergoes spectral change upon inositol hexaphosphate binding to the beta-subunits. In methemoglobin A the spin state and paramagnetic susceptibility increase only in the neutral and alkaline pH ranges upon inositol hexaphosphate binding (Gupta, R.K. and Mildvan, R.S. (1975) J. Biol. Chem. 250, 246; Perutz, M.F., Sanders, J.K.M., Chenery, D.H., Noble, R.W., Penelly, R.R., Fung, L.W.-M., Ho, C., Giannini, I., Porschke, D. and Winkler, H. (1978) Biochemistry 17, 3640). Therefore the hydroxymet form of the alpha-hemes which is responsible for the observed spectral changes must also be responsible for these increases in the magnetic properties of methemoglobin A. Inositol hexaphosphate can bind to methemoglobin at alkaline pH if the beta-hemes are in the high spin form.

Functional and conformational properties of mouse hemoglobins.

Hemoglobins from four strains of mice (C3H/SW, DBA/2J, C57BL6/Kh and A.TH) examined showed pH-dependent heme-heme interactions. The oxygen affinity and cooperativity are reduced at acidic pH. The oxygen equilibrium parameters increase as a function of increasing pH and at physiological pH values they are similar to the corresponding values of human hemoglobin A. The nitrosyl derivatives of these mouse hemoglobins undergo a quaternary structural transition to the T state in going from pH 7.0 to 6.0. These functional and conformational properties are indicative of destabilised oxy structures of mouse hemoglobins at acidic pH. This study also confirms that the cysteine residue at beta 13(A10) position has no influence on the oxygen equilibrium properties or conformation of the molecule.