The major achievements of our laboratory have been in the following areas:
- Mitochondrial cytochrome P450 systems involved in cholesterol conversion to pregnenolone and biosynthesis of other steroid hormones
1.1. Gene and protein structure of P450 system enzymes
1.2. Trophic hormone regulation of gene expression in adrenal cortex
1.3. Regulation of the activity of P450 system enzymes
- Structures of NAD(P) dependent flavoproteins
- Molecular genetics of pseudohypoaldosteronism (PHA)
- Gene and protein structure of cytoskeletal proteins
1. Characterization of the mitochondrial cytochrome P450 systems involved in steroid hormone biosynthesis
The mitochondrial multi-enzyme cytochrome P450 systems catalyze the first regulatory step in steroid hormone biosynthesis in all steroidogenic tissues (the conversion of cholesterol to pregnenolone), and C-11-hydroxylation of adrenal corticosteroids in the adrenal cortex only. These systems include three enzymes: adrenodoxin reductase, adrenodoxin, and a cytochrome P450 (P450scc - EC 188.8.131.52, or P450c11 - EC 184.108.40.206) which constitute an electron transport chain similar to the mitochondrial oxidative phosphorylation chain. During Ph. D. work I developed purification procedures for these enzymes and characterized their kinetic interactions to elucidate their membrane organization and behavior. Our current work continues in this field and concentrates on the structural and functional analysis of these enzymes and molecular biology of their regulation by trophic hormones.
1.1. Gene and protein structure of P450 system enzymes:
- Crystallization and structural analysis of adrenodoxin reductase (in collaboration with Dr. Georg Schulz and Clemens Vonrhein).
- Isolation of the first cDNA for adrenodoxin reductase (NADPH : adrenal ferredoxin reductase) and determination of its sequence (for important implications of this see 2 below).
- Demonstration that adrenodoxin reductase is encoded by a single gene, thus establishing that the different mitochondrial P450 systems are dependent on the same reductase for electron transfer.
- Isolation of the first cDNA for an olfactory epithelium specific microsomal cytochrome P450 (in collaboration with Dr. D. Lancet, Weizmann Institute).
- Isolation of the human cDNA for adrenodoxin reductase and localization of its gene to chromosome 17cen-q25 (in collaboration with Dr. W. L. Miller, University of California-San Francisco).
1.2. Trophic hormone regulation of gene expression in adrenal cortex:
- Cloning the cDNA for the bovine ACTH receptor.
- Demonstration that ACTH induces mitochondrial genome transcription and increases oxidative phosphorylation enzyme cytochrome oxidase activity. This process may be a vital link in ACTH regulation of energy production and reducing equivalent supply for the mitochondrial P450 system enzymes.
- Demonstration that: 1. ACTH can induce all four enzymes of the mitochondrial P450 systems at both mRNA and protein level in adrenal cortex cells. 2. This inductive effect can be achieved by a short duration pulse of ACTH, but not by a pulse of agents that increase intracellular cAMP. These novel observations require modification of previous hypotheses that cAMP is the sole second messenger for the induction of enzymes by trophic hormones in steroidogenic cells. The inductive ability of a brief pulse of ACTH indicates that ACTH can rapidly initiate a series of reactions that result in enzyme induction many hours later. The elucidation of the mediators of this response may lead to a discovery of novel mediators of hormone action in cells.
- Determination of the stoichiometry of these enzymes in the adrenal cortex and corpus luteum demonstrating that the full complement of mitochondrial cytochrome P450 system enzymes are regulated coordinately and that their levels can increase in the corpus luteum up to 100 fold over the levels found in the ovary.
1.3. Regulation of the activity of P450 system enzymes:
- Demonstration that the mitochondrial P450 systems can leak electrons, producing oxygen radicals, and that this leakage is greatly inhibited during substrate metabolism. These findings suggest that e- leakage may be controlled by co-regulation of NADPH and cholesterol availability to the mitochondrial P450scc system.
- Production of specific antibodies against the four mitochondrial P450 system enzymes from bovine adrenal cortex. (The antibodies against adrenodoxin reductase were marketed by OXYGENE).
- Characterization of the mechanism of electron transport in the mitochondrial P450 systems: Providing evidence that this system is not organized as a rigid linear array of electron transport proteins; that adrenodoxin transports electrons from adrenodoxin reductase to cytochrome P450 by shuttling between these two enzymes, and that oxidized adrenodoxin competitively inhibits the latter part of this process.
- Elucidation of some of the mechanisms of ionic activation and inhibition of mitochondrial cytochromes P450.
2. Structures of NAD(P) dependent flavoproteins
- Crystallization and structural analysis of adrenodoxin reductase (in collaboration with Dr. Georg Schulz, Clemens Vonrhein and Gaby Ziegler).
- Isolation of the first cDNA for adrenodoxin reductase (NADPH : adrenal ferredoxin reductase) and determination of its sequence.
- Discovery of a single amino acid sequence difference between type I NAD and NADP binding sites (as a result of the analysis of the adrenodoxin reductase sequence) in NAD(P) dependent oxidoreductases. This led to the derivation of a fingerprint sequence for identifying NADP binding enzymes in sequence databases, and a new hypothesis for changing NAD(P) coenzyme specificities by protein engineering, that has been confirmed (Scrutton, Berry, and Perham, Nature 343:38-43, 1990).
3. Molecular Genetics of Pseudohypoaldosteronism
Pseudohypoaldosteronism Type I (PHA) is a hereditary syndrome of salt wasting resulting from unresponsiveness to mineralocorticoid hormone aldosterone. Our work on this disease is a family project! My pediatrician brother first elucidated that PHA is manifested in two clinically and genetically distinct forms affecting either only the kidney or multiple target organs of aldosterone. Whereas the renal form of PHA is milder and is inherited as an autosomal dominant trait, the multi-system form carries a high rate of mortality and is inherited as an autosomal recessive trait. In most steroid hormone resistance syndromes a mutation in the steroid receptor was shown to be responsible for the disease. Thus, in our work we first examined the possible relationship between PHA and aldosterone receptor gene. Upon finding no relationship in the case of multi-system PHA we directed our attention to other candidate genes. The mutation was localized in the sodium channel subunit genes (see publications for the list of collaborators). Our current work concentrates on the identification of mutations in a series of affected families from Israel and from the UK.
- Determination of the structure of the human gene encoding for the beta subunit of the epithelial amiloride sensitive sodium channel
- Identification of the mutations responsible for PHA in patients with multiple system PHA
4. Analysis of gene and protein structure of cytoskeletal proteins
The cytoskeletal network of most mammalian cells includes three major types of filamentous systems: microfilaments, intermediate filaments and microtubules with respective diameters of 6 nm, 8-10 nm and 25 nm. Each of these filamentous systems is assembled from only one or two different subunits. The proteins that form these filaments are all encoded by multigene families the members of which are differentially expressed in different tissues. My post-doctoral work in this field concentrated on the analysis of cDNA and predicted protein sequence and structure of the cytoskeletal keratins which are members of the intermediate filament protein super-family.
- Determination of the first sequences of the two major classes of human epidermal keratins by analysis of cDNA sequences.
- Identification of the structural domains in intermediate filament proteins and proposal of a model for the secondary structure of these proteins which was found to apply to all other intermediate filament proteins with minor modifications.
- Characterization of the differences between the two classes of cytoskeletal keratins and proposing the universally accepted nomenclature for these as Type I and Type II keratins.
- Providing evidence that, 1) among intermediate filament proteins, the secondary structures of the central regions are conserved despite extensive differences in primary structures, 2) the size heterogeneity among intermediate filament proteins is a result of differences in the length of the amino and carboxy terminal ends rather than the structurally conserved central region.
- Isolation and sequencing of a human cytoplasmic actin cDNA (the first vertebrate cytoplasmic actin cDNA sequence). Providing evidence that the fibrous intermediate filament proteins and the globular microfilament proteins are subject to different types of evolutionary forces.