Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-08-25
2001-04-24
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S252330, C435S193000, C435S194000, C435S320100, C435S135000, C536S023200, C536S023500, C514S04400A
Reexamination Certificate
active
06221658
ABSTRACT:
2. FIELD OF THE INVENTION
The present invention relates to the isolation and characterization of two novel diacylglycerol kinase (DAG kinase) isoforms. More specifically, the invention relates to the isolation of DAG kinase &egr;, DAG kinase &zgr;, and an alternatively spliced species of DAG kinase &zgr;, DAG kinase &zgr;-2 expressed in muscle and methods of use thereof.
3. TECHNICAL BACKGROUND
Lipids are molecules that are fundamental to the existence of all living organisms. Lipids are non-polar molecules that are water-insoluble. As such, the term lipids includes a large number of structurally distinct biomolecules, including phospholipids, glycolipids and sterols, like cholesterol.
Lipids have a variety of biological roles. First, lipids are the major component of biological membranes. Like exterior walls of houses, biological membranes are structurally organized barriers which define and separate cells from the environment and other cells. Like interior walls of houses, biological membranes are structurally organized barriers that compartmentalize and organize the cell's intracellular components.
Biological membranes, however, are not impervious walls. Instead, they are highly selective permeable barriers which regulate the quality and quantity of molecules which are allowed to pass through the membrane. The cell membrane, for example, tightly regulates the amount of water, ions and sugar which can pass into the cell.
One class of lipids which is abundant in all biological membranes is phosphoglycerides. Phosphoglycerides are comprised of a glycerol (a three-carbon alcohol) backbone, two fatty acid chains (long hydrocarbon molecules), and a phosphate. The simplest phosphoglyceride that can be formed is phosphatidic acid. Phosphatidic acid has two fatty acid chains esterified to the hydroxy groups at the C-1 and C-2 positions of glycerol, respectively. The C-3 hydroxyl group of glycerol is esterified to phosphoric acid. While phosphatidic acid is not a major component of biological membranes, it is a key intermediate in the formation of structurally related phosphoglycerides such as phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol which in addition have a sugar moiety attached to the phosphate.
Second, fatty acid-containing lipids are an energy source for cells and organisms. Fatty acids in a series of biological reactions are oxidized by certain cells to yield large amounts of energy necessary to carry out essential biological functions. Fatty acids used for fuel are stored as triglycerides, also referred to as triacylglycerols, or neutral fats. Like phosphatidic acids, triglycerides have a glycerol backbone. Rather than having two fatty acid and a phosphate group, however, triglycerides contain three fatty acid chains.
Triglycerides are an efficient way to store large quantities of energy, and thus are the major energy reservoir in humans and other mammals. The complete oxidation of a typical fatty acid yields approximately 9 kcal/g, as compared to about 4 kcal/g for proteins and carbohydrates. Moreover, unlike carbohydrate energy stores, triglycerides are anhydrous (i.e., do not contain water). Consequently, a gram of triglycerides contains more than six times the energy of one gram of carbohydrate. Taken together, triglycerides account for about 80% of all the energy of an average individual.
Finally, lipids participate in cell-cell communication, differentiation and proliferation. Normal development and function in living organisms requires interactions between cells and the molecules in the surrounding environment. One way cells communicate is via molecules, called transmembrane proteins, that span the cell's biological membrane. When the portion of the transmembrane protein which is outside of the cell encounters specific molecules in the surrounding environment, it undergoes structural and conformational changes which triggers a biological cascade inside the cell.
The binding or interaction of a molecule in the environment with a transmembrane protein frequently activates a membrane-bound enzyme called phospholipase C. The activation of phospholipase C is at the center of many major biological events. For example, the activation of phospholipase C is correlated with cell proliferation. Vasopressin, prostaglandin F2, and bombesin which stimulate cell proliferation stimulate phospholipase C. In addition, phospholipase C plays a role in activation of T lymphocytes of the immune system and fertilization of eggs.
Phospholipase C exerts its biological effects by catalyzing a reaction which cleaves the sugar moiety of the cell membrane lipid phosphatidylinositol 4,5 bisphosphate. The reaction releases diacylglycerol (DAG) and inositol triphosphate. Diacylglycerol and inositol triphosphate, referred to as second messengers, in turn, activate other molecules within the cell. Diacylglycerol, for example, activates an enzyme called protein kinase C (PKC) which is central to numerous biological processes, including the regulation of cell growth and differentiation.
As illustrated in
FIG. 1
, DAG is at the heart of lipid mediated biological events. Diacylglycerol is a precursor to phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol which are indispensable components of biological membranes. In addition, diacylglycerol is a precursor to triglycerides biosynthesis, and therefore, is central to energy stores of organisms. Finally, diacylglycerol is a second messenger which binds and activates protein kinase C leading to numerous biological events.
The proper regulation of diacylglycerol in cells, therefore, is critical for proper biological function. Abnormally high or low levels of diacylglycerol is predicted to alter the lipid biosynthesis and the activity of enzymes that depend on diacylglycerol, like PKC. As expected, when the phosphatidylcholine biosynthetic pathway is blocked in hepatic tissues as a result of choline deficiency, diacylglycerol levels significantly increase. As a result, choline deficient hepatic tissue demonstrate enhanced PKC activity and a higher incident of spontaneous cancer. Indeed, elevated levels of diacylglycerol have been measured in ras-, sis-, src-, fms-, and erbB-transformed cells which have lost their ability to regulate growth. Similar results are observed when cells are treated with phorbol esters, a compound that, like diacylglycerol, activates PKC.
From the foregoing, it will be appreciated that it would be an advancement in the art to provide means for regulating the intracellular pool of diacylglycerol in cells. It would also be an advantage in the art to provide means for regulating cell proliferation by decreasing the pools of diacylglycerol available to activate PKC. It would be yet another advancement in the art to describe enzymes capable of decreasing the pools of diacylglycerol by converting it to phosphatidic acid. It would be a further advancement in the art to identify and disclose the native DNA sequence of various enzymes which catalyze the conversion of DAG to phosphatidic acid, thereby enabling the production of large quantities of the enzymes and their use in gene therapy. Finally, it would be an advancement in the art to provide methods of detecting the messenger RNA and protein levels of these enzymes in a cell.
Such enzymes and DNA sequences are disclosed and claimed herein.
4. BRIEF SUMMARY OF THE INVENTION
The present invention relates to two novel human DAG kinase isoforms capable of catalyzing the conversion of DAG to phosphatidic acid. The first DAG kinase isoform, DGK&egr;, was isolated from an endothelial cell library. The cDNA is 2.6 kilobases (kb) in length and has an open reading frame of 567 amino acid residues which gives a predicted molecular mass of 64 kDa. DGK&egr; has two distinctive zinc finger domains at its N-terminal region, but lacks the E-F hand motifs found in other mammalian DAG kinases. The DGK&egr; catalytic domain is related to the catalytic domain of other DAG kinases and contains two ATP binding motifs.
DGK&egr; mRNA is expressed predominant
Bunting Michaeline
Prescott Stephen M.
Tang Wen
Topham Matthew
Madson & Metcalf
Nashed Nashaat T.
University of Utah Research Foundation
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