Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Patent
1996-02-05
1999-08-17
Rories, Charles C. P.
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
435 691, 435 701, 435 703, 435 711, 4351723, 435183, 435188, 435189, 4352523, 43525411, 4353201, 435375, 536 232, 536 234, 536 235, C12N 510, C12N 121, C12N 1562, C12N 1579
Patent
active
059393181
DESCRIPTION:
BRIEF SUMMARY
INTRODUCTION
1. Technical Field
The present invention relates generally to fused proteins and to genetic engineering of enzymes by production of polynucleotides and using them to express fusion proteins.
2. Background
Hypercholesterolemia is a common problem, affecting about 25% of Americans, and causing extensive mortality and morbidity. Therapeutic approaches include cholesterol-lowering drugs such as nicotinic acid or mevinolin, adsorption of dietary cholesterol to orally administered resins such as cholestyramine, and dietary modification to reduce dietary intake. Therapy by reduced dietary intake often requires reduction or elimination of red meat from the diet, as meat is a major dietary source of cholesterol. Cells may either synthesize cholesterol de novo from acetate or they may receive it by receptor-mediated endocytosis of Low Density Lipoprotein (LDL). Both the synthesis of cholesterol and the cellular uptake of LDL are tightly regulated, but, aside from small amounts of cholesterol secreted as bile acids, there is no cholesterol disposal pathway. Most cholesterol produced in animals is involved in the synthesis and maintenance of cell membranes; however, about 400 mg/day in humans is lost as bile salts (Vlahcevic et al 1990). Small amounts of cholesterol (30-50 mg/day) are converted to adrenal and gonadal steroid hormones (Carr and Simpson 1981, Gwynne and Strauss 1982). Steroidogenesis is initiated by converting cholesterol to pregnenolone, which is biologically and hormonally inactive, by the P450 cholesterol side-chain cleavage enzyme, ("P450scc") (for review see Miller 1988). In steroidogenic tissues, such as the adrenals, gonads, and placenta, pregnenolone is rapidly converted to biologically active steroids by other, tissue-specific enzymes (Miller 1988). When radio-labeled pregnenolone is administered intravenously, it is metabolized by the liver to pregnanediol, and pregnanediol and its sulfates and glucuronides, are excreted in the urine and are thus do not become substrates for steroid hormone synthesis (Arcos 1964; Berstein and Solomon 1970). Deficient P450scc activity causes lipoid adrenal hyperplasia, a generally lethal disease.
Cytochromes P450 comprise a large group of heme-containing proteins found in many prokaryotes and in apparently all eukaryotes (Nelson et al 1993). P450 enzymes metabolize exogenous drugs, environmental pollutants and toxins, and also metabolize endogenously produced steroids, vitamin D, bile acids, prostaglandins, biogenic amines, and leukotrienes. All P450 enzymes have about 500 amino acids and function as terminal oxidases in an electron-transport chain from NADPH. Vertebrate cytochrome P450 enzymes fall into two broad groups: the Type I ("mitochondrial") enzymes found in mitochondria, and the more abundant Type II ("microsomal") enzymes found in the endoplasmic reticulum. The Type I and II P450 enzymes differ substantially in their degree of amino acid sequence identity (Nelson et al 1993) and they differ categorically in the fashion in which they receive reducing equivalents from NADPH. Type I (mitochondrial) enzymes receive electrons through two intermediates: the flavoprotein ferredoxin reductase (also called adrenodoxin reductase ("AdRed")) and the iron-sulfur protein ferredoxin (also called adrenodoxin ("Adx"). Type II ("microsomal") enzymes receive electrons through the intermediary of a single flavoprotein, termed P450 oxido-reductase ("OR") (Gonzalez 1989; Yamano et al. 1989). Microsomal P450c17 apparently can receive electrons from either OR or cytochrome b.sub.5 (Nakajin et al. 1985). Kumamoto et al. (1989) demonstrated that the N-terminal extension peptide (signal peptide) of bovine mitochondrial P450scc precursor contains sufficient information to target in vitro translated P450scc or adrenodoxin (as an extension peptide-adrenodoxin fusion construct having no P450scc activity) to bovine mitochondria.
Mitochondrial cytochrome P450scc converts cholesterol to pregnenolone by catalyzing three reactions on its single active site: 20.alpha.-hydr
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Black Stephen M.
Harikrishna Jennifer A.
Miller Walter L.
Regents of the University of California
Rories Charles C. P.
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