Biomedical subjects
Publications and source records attributed to B C Chung.
Yeast cells harboring mutant plasmids were incubated with 0.1, 0.15, 0.2, 0.3, 0.4, 0.45, 0.6, or 1.0 mM (14C)-17-hydroxyprogesterone for 1h. The steroids in the media were then separated by thin layer chromatography and counted to calculate enzymatic activities. Wt = wild type enzyme. Pt: patient enzyme.
Three Alu sequences have been identified surrounding the human ferredoxin-encoding genes. Among them, one is located about 1000 bp upstream from the active gene, whereas two others flank the ferredoxin pseudogene, psi FDX3. All these Alu sequences contain poly(A) tails and are flanked by direct repeats, indicating that they arose by RNA-mediated transposition events.
Ferredoxin is an iron-sulfur protein that serves as an electron carrier for the mitochondrial oxidation/reduction system. During the characterization of the human ferredoxin gene, we have identified three Alu sequences surrounding it. When these Alu sequences were compared with others, all three of them are more related to the consensus Alu than the 7SL gene, the progenitor of the Alu family. It suggests that they are members of the modern Alu family. Their sequences differ from the Alu consensus sequence by about 5%, indicating that they were inserted into the chromosome about 35 million years ago.
The first step in the synthesis of steroids is catalysed by cytochrome P-450ssc (cholesterol-side-chain cleavage enzyme). We have investigated the synthesis of this enzyme in three cultured cell lines at the protein and hormone secretion levels. Hormone levels were measured by an enzyme immunoassay using a monoclonal antibody against progesterone. The protein level was detected using polyclonal antibodies directed against a P-450scc fusion protein overproduced in Escherichia coli. Utilizing a bacteriophage T7 promoter expression system, a large amount of human P-450scc fusion protein was produced and easily purified. P-450scc was synthesized in the mouse adrenal tumour cell line Y1 and human choriocarcinoma cell line JEG-3, but not in monkey kidney cell line COS-1. The production of P-450scc in Y1 and JEG-3 cells was stimulated by 8-bromo cyclic AMP, the effect of which was not observed until 6 h after induction and was more pronounced at 24 h. Y1 and JEG-3 cells exhibited a difference in progesterone secretion after induction.
The most common cause of congenital adrenal hyperplasia is deficiency of cytochrome P450c21 (21-hydroxylase), which catalyzes the synthesis of adrenal steroids. We have cloned the human P450c21 cDNA into yeast expression vectors under the control of either the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) promoter or the aldehyde-dehydrogenase (ADH) promoter. P450c21 RNA, protein, and enzyme activity can be detected, indicating that both promoters drive the synthesis of P450c21. The expressed P450c21 catalyzes the conversion of both of its substrates, with Km and Vmax values of 0.33 microM and 280 nmoles/hr.nmole of P450c21 protein for progesterone, and 0.23 microM and 450 nmoles/hr.nmole for 17-hydroxyprogesterone. These kinetic properties are similar to those of human P450c21 expressed in COS-1 cells. The microsomal fraction containing P450c21 exhibited an absorption peak at 450 nm upon binding to CO, demonstrating its hemoprotein nature. The CO-difference spectra indicated that there were about 0.08 nmole P450c21 hemoprotein/mg microsomal protein. Coupling this expression system with site-directed mutagenesis, the Asn-172 mutant of P450c21 had about 20-100 lower Vmax values; yet it retained normal affinity toward both substrates. This mutant protein also exhibited an altered absorbance with a peak at 420 nm rather than at 450 nm.
Steroid 21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH), a common genetic disease. To define the relationship between gene mutations and enzyme deficiency, we generated missense mutations of the 21-hydroxylase cDNA at three different sites and characterized the mutant proteins after expressing them in cultured mammalian and yeast cells. Among them, Ser268 and Val281 have been found to be mutated in CAH patients, whereas Cys428 has been implicated as the heme ligand. Our results show mutations at these sites result in complete, partial, or no loss of the enzymatic activity. All the Cys428 mutants had neither enzymatic activity nor P450 absorption, thus supporting the notion that Cys428 is the heme ligand. All the 268-mutants exhibited the same activity as normal 21-hydroxylase, demonstrating that the clinically observed Ser268----Thr change represents a polymorphism rather than the cause of the enzyme deficiency. The 281-mutants had normal Km but greatly reduced Vmax values that also paralleled the reduction in the heme content, in the order Val281 (normal, 100%) greater than Ile281 (50%) greater than Leu281 (20%) greater than Thr281 (10%). Our findings suggest that the methyl group at the beta-carbon of Val281 is required for heme incorporation and consequently enzymatic activity.
Congenital adrenal hyperplasia (CAH) is a common recessive genetic disease caused mainly by steroid 21-hydroxylase (P450c21) deficiency. Many forms of CAH exist resulting from various mutations of the CYP21B gene. We sequenced CYP21B cDNA from a normal person and its genes from a patient with simple virilizing CAH. When comparing several CYP21B sequences, we found it was polymorphic. In the patient, a single base substitution replaced Ile172 (ATC) with Asn (AAC) in one allele while Arg356 (CGG) was converted to Trp (TGG) in the other. A normal P450c21 cDNA clone was transfected into COS-1 cells to produce 21-hydroxylase activity toward its substrates, progesterone and 17-hydroxyprogesterone. Mutants corresponding to Asn172 or Trp356 mutation were constructed by site-directed mutagenesis of the normal c21 cDNA clone. They failed to produce active enzyme toward either substrate upon transfection into COS-1 cells, demonstrating that these mutations caused CAH. Aligning sequences with other P450s, Ile172 could be located in the membrane anchoring domain and Arg356 in the substrate-binding site of P450c21. Both mutations are present in the CYP21A1P pseudogene, suggesting that they may be transferred from CYP21A1P by gene conversion events.
Ferredoxin is an iron-sulfur protein that serves as an electron transport intermediate for mitochondrial cytochromes P450 involved in steroid, vitamin D, and bile acid metabolism. We cloned and characterized the human ferredoxin gene family, which includes two expressed genes and two pseudogenes. Sequence analysis of this gene family revealed that it encodes only one protein product. The expressed genes were assigned to chromosome 11 and pseudogenes to chromosomes 20 and 21 by identifying single-copy probes from each gene segment and hybridizing them to DNA from rodent-human hybrid cells. The pseudogenes lacked introns and contained numerous mutations, including insertion, deletion, and substitution which rendered them inactive. They were 96% and 85% homologous to the expressed gene, yet they were only 78% homologous with each other. The intronless nature, higher diversity among themselves, and distinct chromosomal location of the pseudogenes suggests that they arose by independent, retroposon-mediated events.
Cytochrome P450c21 (steroid 21-hydroxylase) is a key enzyme in the synthesis of cortisol, whose deficiency is the cause of a common genetic disease, congenital adrenal hyperplasia. We have expressed P450c21 (steroid 21-hydroxylase) in E. coli and mammalian cells. In E. coli, P450c21 cDNA was cloned into a T7 expression vector to produce a large amount of P450c21 fusion protein, which enabled antiserum production. In mammalian cells, a plasmid containing full-length P450c21 cDNA (phc21) was constructed and transfected into COS-1 cells to produce active P450c21, which was detected by immunoblotting and 21-hydroxylase activity assay. This system was used to assay mutations involved in the disease. Ile172 of phc21 corresponding to the site of mutation in some cases of the disease was mutagenized to become Asn, Leu, His, or Gln. Mutant as well as normal P450c21 was produced when their cDNAs were transfected into COS-1 cells. The mutant proteins, however, had greatly reduced 21-hydroxylase activities. Therefore, missense mutation at Ile172 resulted in inactivation of the enzyme, but not in repression of enzyme synthesis. The Leu for Ile substitution at amino acid 172 did not result in partial restoration of enzymatic activity, indicating that hydrophobicity at this residue may not play a role in its function.
Congenital adrenal hyperplasia (CAH) is a heterogeneous group of hereditary diseases characterized by deficient adrenal cortisol synthesis. Most CAH is due to 21-hydroxylase (C21) deficiency. Genomic DNA from several families with 21-hydroxylase deficiency and normal controls was analyzed by the Southern blot hybridization technique. The restriction fragment length polymorphism (RFLP) patterns using several endonucleases, such as Taq I, Eco RI and Pvu II, at the C21 gene locus showed a very low frequency of variability in normals and most of the patients with CAH. One proband with CAH lacked the characteristic 3.7 kb Taq I fragment probed with C21 cDNA. This may be due to gene conversion and/or deletion events in the functional C21 gene locus. On the other hand, genes closely linked to the C21 locus such as C4 and HLA-DR are highly polymorphic. Using these flanking genes as probes, it is easy to perform linkage analysis and identify the inheritance trait. Prenatal diagnosis will be possible in these affected families when an additional pregnancy is expected.
The first step to the synthesis of all steroids is catalyzed by P450scc. We constructed nine deletion mutants of the 5'-region of the P450scc gene and connected them to a CAT reporter gene to assay transcriptional activity of the P450scc promoter. A short 145 bp fragment stimulated transcription by two fold. This DNA was active in all cells tested irrespective of their tissue origin and steroidogenic activity. DNA at -145/-573 of the upstream region did not increase transcription any further. DNA including 2500 bp of the upstream region stimulated transcription by 10 fold only in adrenal Y-1 cells. Hence in the -145 region contains a low level P450scc promoter and the 2500 bp DNA possesses an adrenal specific enhancing element.
Adrenodoxin is an iron-sulfur protein serving as an electron transfer intermediate in the mitochondrial cytochrome P450 system. To study its transcriptional regulation we construct a human adrenodoxin genomic clone which includes 333 bp of DNA upstream of the mRNA start site. This DNA contains a TATA box and two GC boxes. When we place it in front of the CAT reporter gene and transfect it into the recipient cell lines, it directs transcription of the CAT gene. This DNA in reverse orientation does not show any transcriptional activity. This promoter element functions in three mammalian cell lines: JEG-3, COS-1, and Y-1.
Adrenodoxin is an iron-sulfur protein serving as an electron transport intermediate for two mitochondrial steroidogenic cytochromes P450. We have cloned and sequenced three human adrenal adrenodoxin cDNAs. The longest 5'-untranslated region was 131 bases long, and the coding sequences, identical in all three clones, predict a preprotein of 180 amino acids. The 3'-untranslated regions were 235, 596, and 776 bases long due to the presence of alternate polyadenylation sites. RNA transfer blots showed multiple size species of adrenodoxin mRNA consistent with finding multiple polyadenylation sites. Similar sized cross-hybridizing RNA species are found abundantly in the adrenal and testis and to a lesser degree in RNA from human fetal brain, spleen, placenta, kidney, liver, and intestine, as well as in cultured fibroblasts, suggesting the same or a very similar iron-sulfur protein is found in mitochondria of nonsteroidogenic tissues. JEG-3 cells, a transformed progesterone-producing line of trophoblastic origin, accumulate mRNAs for cytochrome P450scc (the mitochondrial cholesterol side-chain cleavage enzyme), adrenodoxin, and the fos oncogene when stimulated with 8-bromo-cyclic AMP. Addition of actinomycin D to such cultures blocked cAMP-induced accumulation of mRNAs for cytochrome P450scc and adrenodoxin. Addition of cycloheximide or puromycin to such cultures substantially reduced basal levels and markedly attenuated the cAMP-induced accumulation of cytochrome P450scc mRNA, but augmented the accumulation of adrenodoxin and fos mRNAs in additive and multiplicative fashions, respectively. These data indicate that the cAMP-induced synthesis of the steroidogenic machinery is not wholly dependent on cycloheximide-sensitive protein mediators.
Adrenodoxin is an iron-sulfur protein that serves as an electron transport intermediate for all mitochondrial forms of cytochrome P450. To facilitate studying the regulation of adrenodoxin, we have cloned and determined the structure of the human adrenodoxin gene. It spans more than 20 kb, containing four exons and three introns. The first exon encodes the 60-amino-acid signal peptide, directing transport of the protein into the inner mitochondrial matrix. The mature peptide of 124 amino acids is encoded by the other three exons. The third exon encodes the portion of the protein containing the iron-sulfur center and a domain which binds other components of the electron transport chain. The transcriptional start sites were determined by primer extension and S1 nuclease mapping. The 5'-flanking region of this gene contains canonical promoters including a TATA box at nucleotide position -30 and two GC boxes at nucleotide positions -60 and -100. The sequence at nucleotides -234 to -252 is also highly homologous to the glucocorticoid-responsive element and the estrogen-responsive element.
Adrenodoxin is a small iron/sulfur protein serving as an electron-transport intermediate for all mitochondrial forms of cytochrome P450. Southern blots of normal genomic DNA cleaved with six restriction endonucleases probed with full-length human adrenodoxin cDNA revealed complex patterns indicating the presence of multiple adrenodoxin genes. Southern blots of DNA from a panel of mouse/human somatic cell hybrids identified cross-hybridizing adrenodoxin DNA in two loci, chromosome 11q13----qter and chromosome 20cen----q13.1. Examination of adrenodoxin clones from a genomic DNA library in phage lambda revealed some clones bearing gene fragments interrupted by introns and other clones bearing processed pseudogenes. By probing the mouse/human hybrids with unique intronic DNA and by correlating restriction maps of the phage clones with that of uncloned genomic DNA, we show that the authentic transcribed adrenodoxin gene lies on chromosome 11, while pseudogenes lie on chromosome 20.
Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P450XXIA2 gene encodes cytochrome P450c21 [steroid 21-monooxygenase (steroid 21-hydroxylase), EC 1.14.99.10], which mediates 21-hydroxylation. The P450XXIA2 gene may be distinguished from the duplicated P450XXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P450XXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. We have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAH. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, we conclude that the P450XXIA2 gene "deletions" widely reported in CAH patients probably represent gene conversions, unequal crossovers, or polymorphisms rather than simple gene deletions.
P450c17 is the single enzyme mediating both 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. We sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17. Comparison of the amino acid sequence of P450c17 with two other human steroidogenic cytochromes P450 show much greater homology with P450c21 (28.9%), another microsomal enzyme, than with P450scc (12.3%), a mitochondrial enzyme.
P-450c21, a cytochrome P-450 enzyme [steroid 21-monooxygenase (steroid 21-hydroxylase), EC 1.14.99.10], mediates the 21-hydroxylation of glucocorticoid and mineralocorticoid hormones in the adrenal gland. The complete sequence of a bovine P-450c21 gene shows it is 3447 base pairs long and contains 10 exons. The intron/exon organization and encoded amino acid sequence indicate that P-450c21 represents a unique family of genes in the P-450 gene superfamily. Primer extension and S1 nuclease protection experiments identified several cap sites for initiation of transcription; the principal cap site produces mRNA with a 5' untranslated region only 11 bases long. S1 nuclease protection experiments confirm that P-450c21 is actively expressed in the adrenal and the testis, an organ not known to secrete 21-hydroxylated steroids.