Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-06-16
2004-12-28
Rao, Manjunath (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S004000, C435S006120, C435S069100, C435S183000, C435S252300, C435S320100, C435S325000, C536S023200, C536S023400, C536S023700
Reexamination Certificate
active
06835563
ABSTRACT:
TECHNICAL FIELD OF INVENTION
The present invention relates to novel ABC1 polypeptides and nucleic acid molecules encoding the same. The invention also relates to recombinant vectors, host cells, and compositions comprising ABC1 polynucleotides, as well as to methods for producing ABC1 polypeptides. The invention also relates to antibodies that bind specifically to ABC1 polypeptides. In addition, the invention relates to methods for increasing cholesterol efflux as well as to methods for increasing ABC1 expression and activity. The present invention further relates to methods for identifying compounds that modulate the expression of ABC1 and methods for detecting the comparative level of ABC1 polypeptides and polynucleotides in a mammalian subject. The present invention also provides kits and compositions suitable for screening compounds to determine the ABC1 expression modulating activity of the compound, as well as kits and compositions suitable to determine whether a compound modulates ABC1-dependent cholesterol efflux.
BACKGROUND OF THE INVENTION
Circulating lipids in human plasma or lymphatic fluid consist of cholesterol, cholesteryl esters, triglycerides and phospholipids. These lipids are transported in large molecular complexes called lipoproteins, which consist of a core of cholesteryl esters and/or triglycerides, an envelope of phospholipids and free cholesterol, and apolipoproteins (Scriver et al., Eds., the.
Metabolic and Molecular Basis of Inherited Disease,
7
th
. Ed., p.1841-1851 (McGraw-Hill, New. York 1995)). Apolipoproteins are involved in the assembly and secretion of the lipoprotein, as well as the activation of lipoprotein modifying enzymes, such as lecithin cholesterol acyl transferase (LCAT). In addition, apolipoproteins provide structural integrity and are ligands for a large spectrum of receptors and membrane docking proteins. The plasma lipoproteins are categorized into five types according to size: chylomicrons (largest in size and lowest in density)+very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoprotein (HDL).
Chylomicrons, VLDLs, IDLs, and LDLs transport exogenous and endogenous cholesterol and triacylglycerols to peripheral sites, where the lipids play a role in various metabolic pathways and serve as a major constituent of cell membranes. Chylomicrons are assembled in the intestinal mucosa as a means to transport dietary cholesterol and triacylglycerols to various tissues. VLDLs are formed in the liver to transport endogenous cholesterol and triacylglycerols synthesized by the liver to extra-hepatic tissues, such as muscle and adipose tissue. In fasting serum, VLDLs contain 10-15% of the total serum cholesterol and most of the triglyceride. In circulation, VLDLs are converted to LDLs through the action of lipoprotein lipase. LDLs are the primary plasma carriers of cholesterol for delivery to all tissues, typically containing 60-70% of the total fasting serum cholesterol.
In contrast, HDLs are involved in “reverse cholesterol transport”, the pathway by which excess cholesterol is transported from peripheral sites back to the liver, where it is excreted in the form of bile salts (Glomset, J. A.,
J. Lipid. Res.,
9, 155-167 (1968)). Nascent HDLs are synthesized de novo in the liver and small intestine, as protein-rich disc-shaped particles devoid of cholesterol and cholesterol esters. In fact, a major function of HDLs is to act as circulating stores of apolipoproteins, primarily apo C-I, apo C-II, and apoE. The nascent or protein-rich HDLs are converted into spherical lipoprotein particles through the accumulation of cholesteryl esters obtained from cellular sources. The HDL normally contain 20-30% of the total fasting serum cholesterol.
According to current theories, the reverse efflux of cellular cholesterol to HDL is mediated through two mechanisms: an aqueous diffusion pathway and an apolipoprotein-mediated pathway. The relative importance of these distinguishable mechanisms depends on the cell type and metabolic state (Oram et al.,
J. Lipid. Res.,
37:2743-2491 (1996); Rothblat et al.,
J. Lipid. Res.,
40:781-796 (1999); Stein et al.,
Atherosclerosis,
144:285-301 (1999)). For many cells, the aqueous diffusion pathway is the principle pathway through which cholesterol efflux occurs (Johnson et al.,
Biochim. Biophys. Acta,
1085:273-298 (1991)). This pathway involves the bidirectional exchange of cholesterol between cell membranes and a lipoprotein acceptor, such as HDL, in the extracellular space through a process of passive transport (Remaley et al.,
Arterioscler. Thromb. Vasc. Biol.,
17:1813-1821 (1997); Rothblat et al.,
J. Lip. Res.,
40:781-796 (1999)). The exchange may occur primarily at surface microdomains known as caveolae (Fielding et al.,
Biochemistry,
34:14288-14292 91995)). Net efflux can be driven by conversion of cholesterol in the exteracellular compartment to cholesteryl ester by the action of LCAT.
Alternatively, in macrophage and fibroblast cells, cholesterol and phospholipid efflux is primarily mediated through apolipoproteins, such as apo A-I, apo A-II, and. Apo E (Remaley, supra (1997); Francis, et al.,
J. Clin. Invest.,
96:78-87 (1995); Vega et al.,
J. Intern. Med.,
226:5-15 (1989); Sakar et al.,
Biochim. Biophys. Acta,
1438: 85-98 (1999); Hara et al.,
J. Biol. Chem.,
266:3080-3086 (1991); Fielding et al.,
J. Lipid. Res.,
38, 1503-1521 (1997); Oram et al., J. Lipid. Res., 37, 2743-2491 (1996)). The process of apolipoprotein-mediated lipid efflux particularly dominates in macrophages and other scavenger cells when they are cholesterol-loaded and/or growth-arrested. Apolipoprotein-mediated efflux is an active transport process that requires the direct interaction of the apolipoprotein with the cell surface, the lipidation of the apolipoprotein, and the subsequent dissociation of the lipid-apolipoprotein particle from the cell (Oram, supra (1996); Mendez, A. J.,
J. Lipid Res.,
38, 1807-1821 (1997); Remaley, supra (1997); Mendez, A. J.,
J. Lipid Res.,
37, 2510-2524 (1996)). Once removed from the cell, the cholesterol-rich HDL particles are transported to the liver and removed from the body as described.
Abnormal lipoprotein function and/or metabolism resulting from genetic defect or as a secondary effect of another disorder can have serious biological consequences. In addition to dietary influences, disorders such as diabetes, hypothyroidism, and liver disease can result in elevated plasma levels of LDL-cholesterol and triglycerides. Elevated levels of LDL-cholesterol and triglycerides have been identified as major risk factors associated with the incidence of coronary heart disease, which is the primary cause of death in the United States and other industrialized nations (Hokanson et al.,
J. Cardiovasc. Risk.,
3:213-219 (1996); The Expert. Panel,
JAMA,
269:3015-3023 (1993)). The accumulation of excess LDL-cholesterol on arterial walls can lead to the formation of atherosclerotic plaques, which play a major role in the development of heart disease. A plaque is believed to form when free radicals released from arterial walls oxidize LDL. According to theory, the oxidized form of LDL triggers an inflammatory response, attracting circulating cells to the site which contribute to the formation of a lipid plaque. Among these are macrophages and other cells that contain scavenger receptors that accumulate cholesterol in an unregulated manner (Brown et al.,
Ann. Rev. Biochem.,
52:223-261 (1986)). Vast stores of internal cholesterol result in conversion to a foam cell phenotype, which is believed to be a major contributor to the development of vascular lesions. As the plaque builds up, the arterial walls constrict, reducing blood flow to the heart.
Interestingly, however, an estimated 60% of heart attacks occur in persons who do not have elevated blood levels of LDL-cholesterol. Of these, an estimated 45% are associated with below average blood levels of HDL-cholesterol, indicating that low HDL-cholesterol level is
Garvin Michael
Lawn Richard M.
Oram John F.
Wade David
CV Therapeutics
Rao Manjunath
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