Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1998-10-22
2003-05-13
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S006120, C435S069100, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06562609
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of this invention is cholesterol regulation.
2. Background
Oxysterols are formed by the hydroxylation of the side chain of cholesterol. This modification renders the sterol more hydrophilic and confers two important biological properties. First, the increased hydrophilicity enhances the ability of the oxysterol to cross membranes and thereby facilitates its movement between intracellular compartments, cells and tissues. Second, oxysterols delivered in ethanol to cultured cells, are potent regulators of the expression of genes involved in sterol and fatty acid metabolism (1,2).
The enhanced solubility of oxysterols is exploited by the body to maintain cholesterol homeostasis. In several tissues and cell types, including the brain, kidney, endothelium, and macrophages, cholesterol is converted into oxysterols that subsequently traverse the plasma membrane and are transported to the liver (3-5). In the liver, they are converted into bile acids by a newly described biosynthetic pathway (6). These bile acids are essential for normal lipid and fat-soluble vitamin metabolism (7).
Oxysterols are both positive and negative regulators of gene expression. As positive effectors, they bind to and activate the nuclear receptor LXR (8), which in turn increases transcription of the cholesterol 7&agr;-hydroxylase gene (9). This activation stimulates the conversion of cholesterol into bile acids (10). Mutation of the LXR gene in mice causes a loss of 7&agr;-hydroxylase gene induction and a buildup of cholesterol in the liver (11). As negative regulators, oxysterols suppress the cleavage of two transcription factors known as sterol regulatory element binding proteins-1 and -2 (SREBP-1 and -2) (12). These proteins are synthesized as inactive precursors in the membrane compartment of the cell. When intracellular cholesterol levels decline, SREBPs are cleaved to release amino-terminal fragments that migrate to the nucleus and activate the transcription of a network of genes involved in cholesterol synthesis and supply (12). This activation in turn restores intracellular cholesterol levels.
Several oxysterols occur naturally, including 25-hydroxycholesterol(cholest-5-ene- 3&bgr;,25-diol), 24-hydroxycholesterol(cholest-5-ene-3&bgr;,24-diol), and 27-hydroxycholesterol(cholest-5-ene-3&bgr;,27-diol) (13). Of these three oxysterols, 25-hydroxycholesterol is the most potent regulator of gene transcription when assayed in vitro (1,2,9,11). Hence, 25-hydroxycholesterol biosynthetic enzymes would provide attractive targets for therapeutic inhibitor development, i.e. novel hypocholesteremic agents: by blocking 25-hydroxycholesterol synthesis, SREBPs remain in their active forms and stimulate expression of the LDL receptor, which in turn extracts LDL from the plasma, lowering serum cholesterol.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to cholesterol 25-hydroxylase polypeptides having cholesterol 25-hydroxylase-specific structure and activity, related polynucleotides and modulators of cholesterol 25-hydroxylase function and serum cholesterol. For example, the subject cholesterol 25-hydroxylase polypeptides and polynucleotides can be used to regulate cholesterol 25-hydroxylase activity, and hence serum cholesterol in a mammalian host. The polypeptides may be recombinantly produced from transformed host cells from the subject cholesterol 25-hydroxylase polypeptide encoding nucleic acids or purified from natural sources such as mammalian cells. The invention provides isolated cholesterol 25-hydroxylase hybridization probes and primers capable of specifically hybridizing with natural cholesterol 25-hydroxylase genes, cholesterol 25-hydroxylase-specific binding agents such as specific antibodies, agonists and antagonists, cholesterol 25-hydroxylase transcriptional regulators, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for cholesterol 25-hydroxylase transcripts), therapy (e.g. cholesterol 25-hydroxylase inhibitors to modulate serum cholesterol) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating natural 25-hydroxylase genes and transcripts, reagents for screening chemical libraries for lead pharmacological agents, etc.).
DETAILED DESCRIPTION OF THE INVENTION
The nucleotide sequence of natural human and mouse genes encoding natural human and mouse cholesterol 25-hydroxylase polypeptides are shown as SEQ ID NOS:1 and 3, their natural transcript cDNAs are SEQ ID NO:1, nucleotides 1-1355 and SEQ ID NO:3, nucleotides 1173-2526, respectively, each with an additional 3′ poly A tail, and their full translates are shown as SEQ ID NOS:2 and 4, respectively.
The cholesterol 25-hydroxylase polypeptides of the invention include fragments of SEQ ID NOS:2 and 4 having human cholesterol 25-hydroxylase-specific amino acid sequence, binding specificity and function. Preferred polypeptides comprise at least 10, preferably at least 15, more preferably at least 25, more preferably at least 35, most preferably at least 50 consecutive residues of SEQ ID NO:2, wherein such polypeptides and/or consecutive residues are not contained in any conceptual translate of murine ESTs AA289153 and AA285796, nor human ESTs AIO81548, WO1328, and N45640. The subject domains provide cholesterol 25-hydroxylase domain specific activity or function, such as cholesterol 25-specific hydroxylase or hydroxylase inhibitory activity, SCAP (28) binding or binding inhibitory activity, and/or cholesterol 25-hydroxylase specific antibody binding or binding inhibitory activity.
Cholesterol 25-hydroxylase-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays, e.g. binding assays. The term binding assay is used generically to encompass any assay, including in vitro, cell-cuture or animal-based assays (e.g. using gene therapy techniques or with transgenics), etc. where the molecular interaction of a cholesterol 25-hydroxylase polypeptide with a specific binding target is evaluated. The binding target may be a natural intracellular binding target such as a cholesterol 25-hydroxylase substrate, a cholesterol 25-hydroxylase regulating protein or other regulator that directly modulates cholesterol 25-hydroxylase activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or an cholesterol 25-hydroxylase specific agent such as those identified in screening assays such as described below. Cholesterol 25-hydroxylase-binding specificity may be assayed by hydroxylase activity, hydroxylase activity inhibition (e.g. ability of the subject polypeptides to function as negative effectors in cholesterol 25-hydroxylase-expressing cells), by binding equilibrium constants (usually at least about 10
7
M
−1
, preferably at least about 10
8
M
−1
, more preferably at least about 10
9
M
−1
), by immunogenicity (e.g. ability to elicit cholesterol 25-hydroxylase specific antibody in a heterologous host such as a mouse, rat, goat or rabbit), etc.
In a particular embodiment, the subject polypeptides provide cholesterol 25-hydroxylase-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, the subject polypeptides are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of cholesterol 25-hydroxylase-specific antibodies is assayed by solid phase immunosorbant assays using immobilized cholesterol 25-hydroxylase polypeptides of SEQ ID NOS:2 and 4, see, e.g. Table 1.
TABLE 1
Immunogenic cholesterol 25-hydroxylase polypeptides eliciting
cholesterol 25-hydroxylase-specific rabbit polyclonal antibody:
cholesterol 25-hydroxylase polypeptide-KLH conjugates
immmunized per protocol described above.
Cholesterol 25-hydroxylase
Immuno-
Cholesterol 25-hydroxylase
Immuno-
Polypeptide Sequence
genicity
Polypeptide Sequence
Lund Erik G.
Russell David W.
Achutamurthy Ponnathapu
Board of Regents , The University of Texas System
Moore William W.
Osman Richard Aron
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