Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-01-17
2003-04-29
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C435S004000, C435S006120
Reexamination Certificate
active
06555323
ABSTRACT:
BACKGROUND OF THE INVENTION
ABCA transporters, the largest and most diverse family of transport proteins, are associated with many important biological processes, as well as with clinical problems such as cystic fibrosis, antigen presentation, and multidrug resistance of cancers. The designation ABCA transporters recognizes a highly conserved ATP-binding cassette, which is the most characteristic feature of this super family. Typically, ABCA transporters utilize the energy of ATP hydrolysis to pump substrate across the membrane against a concentration gradient. Each ABCA transporter is relatively specific for a given substrate. Nevertheless, the variety of substrates handled by different transporters is enormous: ABCA transporters specific for amino acids, sugars, inorganic ions, polysaccharides, peptides, and even proteins have been characterized (1).
ABCA1 transporters are implicated in the vectorial movement of a wide variety of substrates across biological membranes (2,3). Most of the mammalianABCA transporters identified so far have been associated with clinically relevant phenotypes (3). Human P-glycoprotein confers resistance to chemotherapeutic drugs on tumor cells (4). Persistent hyperinsulinemic hypoglycemia of infancy is associated with mutation of SUR, the receptor for sulfonylureas (5). Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel (6,7).
The basic structural unit of an ABCA transporter consists of a pair of nucleotide binding folds (NBF) and two transmembrane domains, each composed typically of six transmembrane spanners (2,3). Their activity as transporters is dependent on their interaction with ATP at the NBFs followed by its hydrolysis (8-17), and in some cases evidence has been provided for a further regulation via phosphorylation of serine/threonine residues in the region linking the two symmetric halves (7, 18-20). The NBF domains contain the highly conserved phosphate binding loop (21) that forms intimate contacts with the &bgr;- and &ggr;-phosphates of bound ATP (22) and an additional diagnostic motif, the active transport signature, whose function is so far unknown. The above description is not fully applicable to the newly discovered ½-transporters, though much is analogous.
Low serum HDL cholesterol (HDL-C) concentrations have been identified as a good predictor for coronary artery disease (23,24). A variety of factors contribute to low HDL-C levels including genes harboring a basic defect, modifying genes, and environmental factors (25). Low HDL syndromes are genotypically heterogeneous and understanding their molecular basis could explain the essential role of HDL in plasma cholesterol homeostasis and atherosclerosis.
A major cardioprotective activity of HDL is ascribed to its role in reverse cholesterol transport, which is the flux of cholesterol from peripheral cells such as tissue macrophages, through plasma lipoproteins to the liver, where it can be excreted in the form of bile salts (26). Lipid-poor particles, particularly those containing the major HDL protein, apoA-I, play a major role in this process (27,28). They interact with the cell surface to remove excess cholesterol and phospholipids by an active transport pathway involving the Golgi apparatus (28-31). Although the cellular proteins remain to be identified, recent studies have shown that this pathway is severely impaired in subjects with homozygous Tangiers disease (TD).
TD is a rare genetic disorder that is characterized by near or complete absence of circulating HDL and by the accumulation of cholesteryl esters in many tissues, including tonsils, lymph nodes, liver, spleen, thymus, intestine, andSchwann cells (32,33). Most patients were initially identified by enlarged yellow-orange tonsils and symptoms of neuropathy (33). In addition to zero or near zero plasma levels of HDL, patients with TD have a roughly 50% reduction in LDL and a moderate elevation in triglycerides. Although low levels of HDL represent a clear predictor of premature coronary artery disease, the presence of increased cardiovascular disease in patients with TD was at first unclear, as concomitant reduction in LDL may offer some protection from coronary artery disease. However, a review of 54 cases of homozygous TD revealed a 4- to 6-fold increase in cardiovascular disease compared with controls, depending on the age group considered (34).
Cells from subjects with TD are defective in the process of apolipoprotein-mediated removal of cholesterol and phospholipids (30, 35-37). Thus, it is likely that the severe HDL deficiency in TD stems from the inability of nascentapoA-I to acquire lipids. Because they do not mature into lipid-rich HDL, the nascent lipoproteins in these patients are rapidly removed from the plasma, resulting in near zero levels of circulating HDL and apoA-I (38). Although some cell types can rid themselves of substantial amounts of excess cholesterol by other means such as aqueous diffusion (39), a defect in the pathway of apolipoprotein-mediated efflux is likely to be at the root of the massive tissue deposition of sterols and the pathology observed in patients with TD (40).
Using linkage analysis and positional cloning, three separate groups identified mutations in the human ABCA1 gene that are linked to Tangiers disease (41-43). ABCA1 possesses all the distinguishing features of other ATP-binding proteins, including two ATP-binding segments and two transmembrane domains (44). ABCA1 is a 220-kDa glycoprotein expressed by macrophages and required for engulfment of cells undergoing programmed cell death (45). Additionally, ABCA1 has been found in numerous other cells types (46). ABCA1 is activated by protein kinases (45) and is modulated at the transcriptional level by increased cellular cholesterol stores (47). Recently, ABCA1 has been associated with the initial steps of reverse cholesterol transport from cells (48). In addition, ABCA1 may have critically important functions in the body by virtue of its ability to function as a cholesterol gatekeeper.
According to Becq et al. (45), ABCA1 generates an anion flux sensitive to glibenclamide, sulfobromophthalein, and blockers of anion transporters. The anion flux generated by ABCA1 is up-regulated by orthovanadate, cAMP, protein kinase A, okadaic acid, and other compounds. In other ABCA transporters, mutating the conserved lysine in the nucleotide binding folds was found to severely reduce or abolish hydrolysis of ATP, which in turn altered the activity of the transporter. In ABCA1, replacement of the conserved lysine 1892 in the Walker A motif of the second nucleotide binding fold increased the basal ionic flux, did not alter the pharmacological inhibitory profile, but abolished the response to orthovanadate and cAMP agonists. Therefore, it was concluded that ABCA1 is a cAMP-dependent and sulfonylurea-sensitive anion transporter (45).
SUMMARY OF THE INVENTION
In a first aspect, the present invention relates to a method of determining the ability of a test compound to affect the activity of ABCA1 protein, said method comprising the steps of introducing labeled substrate into ABCA1-expressing cells, adding a test composition comprising said test compound to a first portion of said cells, and adding a control composition to a second portion of said cells, wherein said control composition is essentially identical to said test composition except that said control composition does not include said test compound, and comparing the level of efflux of substrate from said first portion of said cells to the level of efflux of substrate from said second portion of said cells, wherein a change in the level of efflux indicates that said test compound affects the activity of the ABCA1 protein.
In a preferred embodiment of the first aspect, a positive control composition is added to a third portion of said cells, said positive control composition being essentially identical to said control composition except that said positive control composition
Bamberger Mark J.
Francone Omar L.
Benson Gregg C.
Huff Sheela
Pfizer Inc.
Raymer Gregory P.
Richardson Peter C.
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