The human GPI1 gene is required for efficient glycosylphosphatidylinositol biosynthesis.

The first step in glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis that is defective in paroxysmal nocturnal haemoglobinuria is mediated by an N-acetylglucosaminyl transferase expressed in the endoplasmic reticulum. Six human genes encode subunits of this enzyme, namely PIG-A, PIG-C, PIG-H, PIG-P, GPI1, and DPM2. Here, the human GPI1 gene is characterised. This gene is organised into eleven exons. The locus was mapped to chromosome 16p13.3 near the haemoglobin alpha chain locus. GPI1 is expressed ubiquitously in human cells and tissues. Expression levels are markedly elevated in haematopoietic tissues (bone marrow, foetal liver). To determine whether human GPI1 is essential for human GPI biosynthesis, antisense RNA was expressed in HEK293 cells. Transfectants exhibited a marked but incomplete decrease in the expression of a GPI-linked reporter protein, confirming that GPI1 is required for efficient GPI biosynthesis. In contrast, expression of GPI-linked proteins is normal in lymphatic cell lines from individuals with the alpha thalassaemia/mental retardation syndrome, which is characterised by large deletions from chromosome 16p removing one of the two GPI1 alleles along with the haemoglobin alpha locus. In conclusion, GPI1 plays an important role in the biosynthesis of GPI intermediates. Due to its autosomal localisation, the heterozygous deletion of GPI1 does not lead to an overt defect in the expression of GPI-linked proteins.     

Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity.

In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11beta-hydroxylase [Bülow, H.E.,M?bius, K., B?hr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res. Commun. 221, 304-312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate specificity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone-specific synthesis of mineralocorticoids and glucocorticoids, respectively. Thus, CYP11B2 displayed a fourfold higher ability to perform 11beta-hydroxylation of androstenedione than CYP11B1, while this difference is diminished with the size of the C17 substituent of the substrate. Furthermore, analyses with the electron transfer protein adrenodoxin indicate differential sensitivity of CYP11B1 and CYP11B2 as well as the three hydroxylation steps catalysed by CYP11B2 to the availability of reducing equivalents. Together, both mechanisms point to novel protein intrinsic modalities to achieve tissue-specific production of mineralocorticoids and glucocorticoids in the guinea pig. In addition, we conducted phylogenetic analyses. These experiments suggest that a common CYP11B ancestor gene that possessed both 11beta-hydroxylase and aldosterone synthase activity underwent a gene duplication event before or shortly after the mammalian radiation with subsequent independent evolution of the system in different lines. Thus, a differential mineralocorticoid and glucocorticoid synthesis might be an exclusive achievement of mammals.     

The art gene product of human immunodeficiency virus is required for replication.

A mutation that alters the initiation codon of the art gene of the human immunodeficiency virus renders the virus replication defective. The replication defect of this mutation can be complemented in trans by the art gene product. These findings indicate that the art protein is essential for replication and, as such, represents a good target for development of new antiviral agents that could be useful in the control of human immunodeficiency virus infection.     

The human steroid hydroxylases CYP1B1 and CYP11B2

Major advances have been made during the last decade in our understanding of adrenal steroid hormone biosynthesis. Two key players in these pathways are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2, which catalyze the final steps in the biosynthesis of cortisol and aldosterone. Using data from mutations found in patients suffering from steroid hormone-related diseases, from mutagenesis studies and from the construction of three-dimensional models of these enzymes, structural information could be deduced that provide a clue to the stereo- and regiospecific steroid hydroxylation reactions carried out by these enzymes. In this review, we summarize the current knowledge on the physiological function and the biochemistry of these enzymes. Furthermore, the pharmacological and toxicological importance of these steroid hydroxylases, the means for the identification of their potential inhibitors and possible biotechnological applications are discussed.     

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11β-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC₅₀ values of 1.6 ± 0.1 and 9.9 ± 0.9 nM (mean ± SEM, n = 3–6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K_i values of 0.8 ± 0.04 and 2.2 ± 0.2 nM (n = 3–4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.     

Congenitally defective aldosterone biosynthesis in humans: the involvement of point mutations of the P-450C18 gene (CYP11B2) in CMO II deficient patients.

The gene for steroid 18-hydroxylase (P-450C18) has been recently assigned to encode corticosterone methyl oxidases Type I and Type II which were previously postulated to catalyze the final two steps in the biosynthesis of aldosterone in humans. Molecular genetic analysis of the P-450C18 gene is three patients from three different families affected with CMO II deficiency has indicated that a point mutation of CGG----TGG (181Arg----Trp) in exon 3 and one of GTG----GCG (386Val----Ala) in exon 7 occur exclusively in the gene of the patients. Analysis of PCR products by restriction enzymes (HapII and HphI) has indicated that the patients are homozygous and the unaffected parent is heterozygous for both mutations, in accordance with the established concept that CMO II deficiency is inherited in an autosomal recessive manner. These data clearly provide the molecular genetic basis for the characteristic biochemical phenotype of CMO II clinical variants.     

Mechanisms transducing the aldosterone secretagogue signal of endothelins in the human adrenal cortex

Evidence has been provided that the 21-amino acid hypertensive peptide endothelin (ET)-1 exerts a potent secretagogue effect on human adrenocortical zona glomerulosa (ZG), acting through two receptor subtypes, called ET(A) and ET(B), the signaling mechanism(s) of which has (have) not yet been investigated. Collagenase dispersed human ZG cells were obtained from normal adrenals of patients undergoing nephrectomy/adrenalectomy for renal cancer. The selective ET(A)- and ET(B)-receptor activation was obtained by exposing dispersed cells to ET-1 plus the ET(B)-receptor antagonist BQ-788 and to the ET(B)-receptor agonist BQ-3020, respectively. The phospholipase (PL) C inhibitor U-73122 abolished ET(A) receptor-mediated secretory response, but only partially prevented the ET(B) receptor-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin, the calmodulin inhibitor W-7 and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes. When added together, calphostin-C and wortmannin or W-7 abolished ET(A)-mediated secretory response, but only decreased ET(B)-mediated one. The ET(B) receptor-, but not the ET(A) receptor-mediated aldosterone response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. ET(A)-receptor activation raised inositol triphosphate (IP(3)) production from dispersed ZG cells, while ET(B)-receptor stimulation enhanced both IP(3) and prostaglandin-E(2) production. Collectively, our findings indicate that ETs stimulate aldosterone secretion from human ZG cells, acting through ET(A) receptors exclusively coupled to PLC/PKC-dependent pathway and ET(B) receptors coupled to both PLC/PKC- and COX-dependent cascades.     

The tnpR gene product of TnA is required for transposition immunity

Summary A mutant of TnA no longer recognizing immune plasmids has been isolated. The mutation is complemented in trans by a functional tnpR gene. The requirement for wild type tnpR gene product for the establishment of transposition immunity was confirmed by the use of a derivative of transposon Tn3 in which both the tnpA and the tnpR genes are partly deleted. This deleted Tn3 was shown to transpose onto an immune plasmid in the presence of a wild type tnpA gene but not in the presence of both tnpA and tnpR genes.     

The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation.

Encapsidation of the pregenomic RNA into nucleocapsids is a selective process which depends on specific RNA-protein interactions. The signal involved in the packaging of the hepatitis B virus (HBV) RNA pregenome was recently defined as a short sequence located near the 5' end of that molecule (Junker-Niepmann et al., EMBO J., in press), but it remained an open question which viral proteins are required. Using a genetic approach, we analyzed whether proteins derived from the HBV P gene play an important role in pregenome encapsidation. The results obtained with point mutations, deletions, and insertions scattered throughout the P gene clearly demonstrate that (i) a P gene product containing all functional domains is required both for the encapsidation of HBV pregenomic RNA and for packaging of nonviral RNAs fused to the HBV encapsidation signal, (ii) known enzymatic activities are not involved in the packaging reaction, suggesting that P protein is required as a structural component, and (iii) P protein acts primarily in cis, i.e., pregenomic RNAs from which P protein is synthesized are preferentially encapsidated.     

Mutation T318M in the CYP11B2 gene encoding P450c11AS (aldosterone synthase) causes corticosterone methyl oxidase II deficiency.

Corticosterone methyl oxidase (CMO) deficiency refers to disorders of aldosterone synthesis due to mutations in the CYP11B2 gene encoding cytochrome P450c11AS, which is the adrenal aldosterone synthase. Type I CMO deficiency is associated with low concentrations of 18OH-corticosterone and aldosterone, due to severe mutations in P450c11AS; while type II CMO deficiency is associated with high concentrations of 18OH-corticosterone and low concentrations of aldosterone, due to less severe mutations of P450c11AS. A single type of mutation, compound homozygosity for R181W and V386A, has been reported as the cause of CMOII deficiency in an inbred population. We now report a patient with a typical clinical and hormonal picture of CMOII deficiency. Direct sequencing of patient and parent DNAs showed that the mother's allele contributed R181W and the deletion/frameshift mutation delta C372, while the father's allele contributed T318M and V386A. These mutants were recreated in cDNA expression vectors singly and in the parental pairs, showing that neither allele contributed any measurable activity. This would suggest the patient should have CMOI deficiency. These studies suggest that other factors besides P450c11AS are involved in the genesis of the distinctive CMOI and CMOII phenotypes.     

Discovery of indazole aldosterone synthase (CYP11B2) inhibitors as potential treatments for hypertension

We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement.     

The Ataxia telangiectasia gene product is required for oxidative stress-induced G1 and G2 checkpoint function in human fibroblasts

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G(1) and G(2) phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage.     

The adrenocortical tumor cell line NCI-H295R as an in vitro screening system for the evaluation of CYP11B2 (aldosterone synthase) and CYP11B1 (steroid-11beta-hydroxylase) inhibitors

Aldosterone plays a key role in salt and water homeostasis but is also involved in the development and progression of congestive heart failure and myocardial fibrosis. As a new pharmacological strategy for the treatment of these diseases, we propose the inhibition of the key enzyme of mineralcorticoid formation, CYP11B2 (aldosterone synthase). For studies of the effects of CYP11B2 inhibitors on the adrenal cortex, we selected the NCI-H295R cell line which expresses most of the key enzymes necessary for steroidogenesis. To evaluate this cell line as a test system for effects and side effects of CYP inhibitors, we established assays using radiolabeled substrates of CYP11B2 and CYP11B1 and subsequently tested a series of CYP11B2 inhibitors including the CYP19 inhibitor fadrozole. Fadrozole and compounds 6, 9 and 10 were more potent towards CYP11B2 compared to CYP11B1 with IC(50) values in the nanomolar range. To analyze their overall effect, the formation of steroids in the cell culture supernatant was monitored. All compounds led to a concentration-dependent reduction of the aldosterone secretion but also reduced the formation of cortisol and androgens. In conclusion, the H295R cell line is a suitable tool for the prediction of overall side effects of CYP11B2 inhibitors on steroidogenesis.     

The maternal gene product D7 is not required for early Xenopus development.

The Xenopus D7 gene codes for a novel protein whose expression is restricted to early development. D7 protein is synthesized for the first time during oocyte maturation (1988, Genes Dev. 2, 1296-1306). Injection of D7 RNA into the full-grown oocyte and its subsequent translation into D7 protein neither induced oocyte maturation nor affected the kinetics of hormone-induced maturation. Overexpression of D7 protein by 20-fold in the early Xenopus embryo by injection of D7 RNA into fertilized eggs did not affect subsequent development. Oocytes specifically lacking D7 mRNA were generated by oligodeoxynucleotide-mediated RNA destruction within the oocyte. Unfertilized eggs generated from such oocytes lacked detectable D7 protein, but nevertheless could be activated and fertilized. Embryos generated from such eggs, estimated to contain less than 5% of wildtype levels of D7 protein, developed normally up to the tailbud stage. Thus the D7 protein, the product of a maternal mRNA that is under strict translational repression in oocytes, appears not to be required for oocyte maturation, activation, fertilization or early embryonic development in Xenopus.     

Development and evaluation of a pharmacophore model for inhibitors of aldosterone synthase (CYP11B2)

Recently, we proposed inhibition of aldosterone synthase (CYP11B2) as a novel strategy for the treatment of congestive heart failure and myocardial fibrosis and synthesized a large number of inhibitors. In this work, a pharmacophore model for CYP11B2 inhibitors was developed by superimposition of active and non-active compounds. This model was confirmed by the synthesis of two pyridyl substituted acenaphthene derivatives (A,B). This new class of compounds as well as the pharmacophore could be helpful for the discovery of novel inhibitors.     

N-(Pyridin-3-yl)benzamides as selective inhibitors of human aldosterone synthase (CYP11B2)

A series of 23 N-(Pyridin-3-yl)benzamides was synthesized and evaluated for their potential to inhibit human steroid-11β-hydroxylase (CYP11B1) and human aldosterone synthase (CYP11B2). The most potent and selective CYP11B2 inhibitors (IC₅₀ values 53-166 nM) were further evaluated for their potential to inhibit human CYP17 and CYP19, and no inhibition was observed. Clear evidence was shown for N-(Pyridin-3-yl)benzamides to be a highly selective class of CYP11B2 inhibitors in vitro.