Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1996-06-07
2001-02-27
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C536S023100, C536S023500
Reexamination Certificate
active
06194555
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to compositions and methods for the regulation of steroidogenesis. More particularly, it concerns compositions and methods relating to the regulation of transport of cholesterol into the mitochondria of a cell for the synthesis of androgens, estrogens, glucocorticoids, mineralocorticoids, and progestagens. The invention also relates to methods for detecting and treating steroid hormone-dependent disorders. The nucleic acid molecules of the present invention also provide methods for screening a sample for steroid hormone-dependent disorders, as well as to methods for preparing recombinant proteins for StAR. The invention further relates to the field of nucleic acid probes and primers, as the various nucleic acid molecules of the invention may be used as molecular probes in all of the aforedescribed methods, as well as primers for amplifying particular sequences of interest.
BACKGROUND OF THE INVENTION
The testis is known to be the source of circulating androgens that are responsible for the maintenance of the secondary sexual characteristics in the male. In most species the testis has two separate compartments: the seminiferous tubules that contain the Sertoli cells, the peritubular cells, and the germ cells; and the interstitial compartment that contains the Leydig cells, macrophages, lymphocytes, granulocytes and the cells composing the blood, nerve and lymphatic structures.
The Leydig cells, located in the interstitial compartment and comprising approximately 2-3% of the total testicular cell number in most species, are the only cells in the testis that contain two key steroidogenic enzymes, namely, cytochrome P450 side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). Thus, Leydig cells are the only testicular cells capable of the first two steps in steroidogenesis; i) the conversion of cholesterol, the substrate for all steroid hormones, to pregnenolone; and ii) conversion of pregnenolone to progesterone. Therefore, the interstitial compartment in general, and the Leydig cells in particular synthesize virtually all of the steroids produced in the testis, with testosterone being the major steroid biosynthesized.
The major stimulus for the biosynthesis of testosterone in the Leydig cell is the gonadotrophic hormone, luteinizing hormone (LH). LH is secreted from specific cells located in the anterior pituitary and it interacts with specific receptors on the surface of the Leydig cell and initiates the signal for testosterone production. Cellular events occur rapidly in response to the trophic hormone stimulation of Leydig cells, and result in the synthesis and secretion of testosterone. These rapid or acute effects of hormone stimulation occur within minutes and can be distinguished temporally from the slower chronic effects that occur on the order of many hours and that involve mechanisms to increase gene transcription and translation of the steroid hydroxylase cytochrome P450 enzymes involved in the biosynthesis of these steroids.
The rate-limiting enzymatic step in steroidogenesis is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage complex (CSCC) which is localized to the mitochondrial inner membrane (Stone and Hechter, 1954; Karaboyas and Koritz, 1965; Simpson, et al. 1972). However, the delivery of the substrate cholesterol from cellular stores and the outer mitochondrial membrane to the inner mitochondrial membrane and the CSCC is the true regulated, rate-limiting step in this process (Crivello and Jefcoate, 1980; Jefcoate, et al., 1987). Cycloheximide, an inhibitor of protein synthesis, blocks the hormone-induced steroid production in two steroidogenic tissues of the rat; the adrenal and testis (Ferguson, 1963; Garren, et al., 1965; Davis and Garren, 1968; Jefcoate et al., 1974; Mendelson et al., 1975; Cooke, et al., 1975). This block is at the point of transfer of cholesterol from the outer to the inner mitochondrial membrane and the CSCC (Simpson et al., 1972; Privalle et al. 1983). Therefore, acute regulation of steroidogenesis requires de novo protein synthesis (Jefcoate et al., 1986).
During protein import into the mitochondrial matrix, the inner and outer mitochondrial membranes become closely associated and form protein translocation “contact sites” (Schleyer and Neupert, 1985; Schwaiger et al., 1987; Glick, et al., 1991). Phospholipids are transferred from the outer mitochondrial membrane to the inner mitochondrial membrane at these membrane “contact sites” (Simbeni et al., 1990; Simbeni et al., 1991; Ardail et al., 1991). Therefore, the intramitochondrial cholesterol translocation required for steroidogenesis may also occur at membrane contact sites. An increase in intrrmitochondrial membrane contacts by a hormone-dependent, cycloheximide-sensitive mechanism may regulate cholesterol transport to the CSCC (Jefcoate, et al., 1986). Thus, in the acute regulation of steroidogenesis, a putative function for the newly synthesized regulatory protein may be to facilitate the formation of mitochondrial contact sites that would result in an increased rate of transfer of cholesterol to the inner membrane and CSCC which ultimately would result in the observed increase in the rate of steroid production. However, the seareh for these cycloheximide-sensitive regulatory protein(s) has been ongoing for nearly 30 years, but, as yet, the mechanism of cholesterol transfer to the CSCC is not known.
The present inventors have previously identified a family of hormone-induced mitochondrial proteins in MA-10 cells that regulate cholesterol delivery to the inner mitochondrial membrane and the CSCC. These proteins have been described as the mitochondrial 37 kDa, 32 kDa, and 30 kDa molecular weight proteins and they are synthesized in response to either LH and hCG or by stimulation with the CAMP analogue, Bt2cAMP (Stocco and Kilgore, 1988). The 30 kDa species consists of four separate proteins and proteolytic digestion of all four forms indicates that they are all modified forms of the same protein (Stocco and Chen, 1991). Pulse chase experiments and tryptic peptide mapping of the 37 kDa and 3OkDa proteins indicated that the 37 kDa form is a precursor to the 30 kDa protein (Stocco and Sodeman, 1991; Epstein and Orne-Johnson, 1991). These reports, however, lack information regarding the structure of the nucleic acid molecules and protein molecules involved in steroidogenesis.
Lipoid congenital adrenal hyperplasia (LCAH) is a lethal autosomal recessive disease that results in a complete inability of a newborn infant to synthesize steroids. The lack of mineralocorticoids and glucocorticoids results in death within days to weeks of birth if not detected and treated with adequate steroid hormone replacement therapy. This condition is manifested by the presence of large adrenals containing very high levels of cholesterol and cholesterol esters and also by an increased amount of lipid accumulation in testicular Leydig cells, though this level is somewhat lower than that seen in adrenals. As isolated, mitochondria from adrenals and gonads of affected patients cannot convert cholesterol to pregnenolone (Camacho et al., 1968; Degenhart et al., 1972; Koizumi et al., Hauffa et al., 1985). The P450scc enzyme that converts cholesterol to pregnenolone has been shown to be nornal in patients who suffered from this disease (Lin et al., 1991). Thus, the defect lies upstream of P450scc at the point of cholesterol delivery to the enzyme.
Prior art lacks sufficient identification of the agent(s) responsible for the LCAH metabolic defect and defects in cholesterol transport, lacks screening methods for their detection, and lacks provision of pharmacological agents effective in alleviating the defects. Because of these problems, known procedures are not completely satisfactory despite efforts of persons skilled in the art, and the present inventors have searched for improvements. Further characterization of agents involved in these defects at the amino acid and nucleic acid levels w
Clark Barbara J.
Stocco Douglas M.
Cox & Smith Incorporated
Riley Jezia
Texas Tech University Health Sciences Center
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