Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-06-08
2003-06-17
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C536S023200, C536S023400, C435S069100, C435S193000, C435S348000, C435S320100, C435S252300
Reexamination Certificate
active
06579974
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of the invention is enzymes, particularly acyltransferases.
2. Background of the Invention
The ability to synthesize sterol esters is fundamental to most eukaryotic cells. Sterol esterification is thought to participate in the maintenance of cell membrane sterols at levels optimal for normal cell function. In mammalian cells, cholesterol esterification is catalyzed by the enzyme acyl CoA:cholesterol acyltransferase (E.C. 2.3.1.26, ACAT). ACAT activity has been implicated in a number of physiologic processes. In the small intestine, ACAT has been proposed to play a role in cholesterol absorption by maintaining a free cholesterol diffusion gradient across the enterocyte surface through the intracellular formation of cholesterol esters. Cholesterol ester formation by ACAT has also been hypothesized to be important for the assembly and secretion of apolipoprotein B-containing lipoproteins in the intestine and the liver. In the adrenal glands and other steroidogenic tissues, ACAT synthesizes cholesterol esters that accumulate in cytosolic droplets where they can serve as cholesterol substrate stores for steroidogenesis. In macrophages, ACAT generates intracellular cholesterol esters that are stored as cytosolic lipid droplets, a characteristic feature of macrophage foam cells in atherosclerotic lesions.
Recent evidence has suggested that more than one ACAT exists in mammals. A human ACAT cDNA was first identified from a macrophage cDNA library. The disruption of the mouse homolog of this ACAT gene (Acact) yielded viable, ACAT-deficient (Acact
−/−
) mice that were characterized by tissue-specific reductions in cholesterol esters. Cholesterol ester stores were markedly reduced in adrenal cortices and cultured peritoneal macrophages; however, substantial levels of ACAT activity were present in Acact
−/−
livers, and intestinal cholesterol absorption was normal, indicating that another ACAT enzyme was active in these tissues. Studies examining the tissue distribution of Acact mRNA expression also supported the hypothesis that more than one ACAT exists, as did previous biochemical and ACAT inhibitor studies showing differences between liver and aorta/macrophage ACAT activities. The above results indicate that a second ACAT enzyme contributes to cholesterol esterification activity in the liver and small. intestine.
As such, there is much interest in the identification, isolation and characterization of this putative second ACAT enzyme.
Relevant Literature
U.S. Pat. No. 5,484,727 reports the cloning of the Human ACAT-1 gene.
Farese, “Acyl CoA:cholesterol acyltransferase genes and knockout mice,” Curr Opin Lipidol (April 1998) 9(2):119-123, provides a review of the current knowledge of ACAT genes.
ACAT-1 is described in Goodman, D. S., Physiol. Rev. (1965) 45: 747-839; Suckling & Strange, J. Lipids Res. (1985) 26:647-671 and Chang et al., Annu. Rev. Biochem. (1997) 66: 613-638; Meiner et al., “Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: evidence suggesting multiple cholesterol esterification enzymes in mammals,” Proc Natl Acad Sci U S A (Nov. 26, 1996) 93(24):14041-14046; Meiner et al., “Tissue expression studies on the mouse acyl-CoA:cholesterol acyltransferase gene (Acact): findings supporting the existence of multiple cholesterol esterification enzymes in mice,” J Lipid Res (September 1997) 38(9):1928-1933; Erickson et al., “Acyl-coenzyme A:cholesterol acyltransferase in human liver. In vitro detection and some characteristics of the enzyme,” Metabolism (October 1980)29(10):991-996; Tabas et al., “Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate,” J Biol Chem (Jan. 25, 1988) 263(3):1266-1272; and Uelmen et al., “Tissue-specific expression and cholesterol regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) in mice. Molecular cloning of mouse ACAT cDNA, chromosomal localization, and regulation of ACAT in vivo and in vitro,” J Biol Chem (Nov. 3, 1995) 270(44):26192-26201.
The role of ACAT in various biological processes is discussed in: Field et al., Gastroenterology (1990) 99:539-551; Wilson et al., J. Lipid Res. (1994) 35:943-955; Dixon & Ginsberg, Annu. Rev. Biochem. (1993) 34:167-179; Brown & Goldstein, Annu. Rev. Biochem. (1983) 52:223-261.
SUMMARY OF THE INVENTION
Nucleic acid compositions encoding novel polypeptide products with acyl CoA:cholesterol acyltransferase activity, as well as the polypeptide products (ACAT-2) encoded thereby and methods for producing the same, are provided. The subject polypeptide and nucleic acid compositions find use in a variety of applications, including research, diagnostic, and therapeutic agent screening applications, as well as in therapeutic applications.
DETAILED DESCRIPTION OF THE INVENTION
Nucleic acid compositions encoding novel polypeptide products with acyl CoA:cholesterol acyliransferase activity, as well as the polypeptide products (ACAT-2) encoded thereby and methods for producing the same, are provided. The subject polypeptide and/or nucleic acid compositions find use in a variety of different applications, including research, diagnostic, and therapeutic agent screening/discovery/preparation applications, as well as in the treatment disease conditions associated with ACAT-2 activity.
Before the subject invention is further described, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
N
UCLEIC
A
CID
C
OMPOSITIONS
Nucleic acid compositions encoding polypeptide products (hereinafter ACAT-2 and described in greater detail below), as well as fragments thereof, are provided. By nucleic acid composition is meant a composition comprising a sequence of DNA having an open reading frame that encodes an ACAT-2 polypeptide, i.e. a gene encoding a polypeptide having ACAT activity, where the encoded polypeptide is not ACAT-1 as disclosed in U.S. Pat. No. 5,484,727, the disclosure of which is herein incorporated by reference, and is capable, under appropriate conditions, of being expressed as an ACAT-2 polypeptide. Also encompassed in this term are nucleic acids that are homologous, substantially similar or identical to the nucleic acids encoding ACAT-2 polypeptides or proteins. Thus, the subject invention provides genes encoding mammalian ACAT-2, such as genes encoding human ACAT-2 and homologs thereof and mouse Acat-2 and homologs thereof.
The coding sequence of the mouse Acat-2 gene, i.e. the mouse cDNA encoding the mouse Acat-2 enzyme, has the nucleic acid sequence identified as SEQ ID NO:01, infra. Acat-2 maps to mouse chromosome 15. The coding sequence of the human ACAT-2 gene, i.e. the human cDNA encoding the human ACAT-2 enzyme, has the nucleic acid sequence identified as SEQ ID NO:02, infra. Of interest in many embodiments is a nucleic acid (or the complement thereof) that hybridizes with either SEQ ID NO.01 or SEQ ID NO. 01 under stringent conditions.
The source of homologous genes to those specifically listed above may be any mammalian species, e.g., primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines, equines, yeast, nematodes, etc. Between mammalian species, e.g. human and mouse, homologs have substantial sequence similarity, e.g. at
Cases Sylvaine
Erickson Sandra K.
Farese, Jr. Robert V.
Bozicevic, Field & Francis
Field Bret E.
Minnifield Nita
The Regents of The University of California
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