Phosphatidylinositol-based probes of membranes, proteins, and enzymes.

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Cells contain areas filled with water and others filled with fats and lipids. Biological membranes, the barriers that surround cells and their internal organelles, are composed of lipids that have very poor solubility in water. There are times, however, when a cell requires that lipids be moved from one place to another. Researchers have discovered proteins that are capable of picking up specific lipids and moving them from one membrane location to another. Many of these proteins, generically termed “lipid transfer proteins”, have been discovered and described over the last 50 years. But their names may have obscured our thinking about the true function of these proteins. In fact, demonstrating the ability of these proteins to transfer lipids in living cells is not trivial. We have made fluorescent probes to follow just this sort of action for the protein that transfers vitamin E around mammalian cells. We have also explored the function of phosphatidylinositol transfer proteins (PITPs) that carry certain phospholipids. These proteins are critical for the survival of yeast, and mutations of the mammalian forms result in a variety of neurological diseases and developmental problems.Mounting evidence from genetic experiments with yeast suggests that PITPs are not just simple transfer proteins but rather molecular helpers of membrane-associated lipid kinase enzymes. Kinases attach phosphate groups to molecules and some lipid kinases produce powerful signaling lipids (essentially acting as cellular GPS signals) called phosphatidylinositol phosphates (PIPs). This concept is a fundamental re-thinking of the role of lipid transfer proteins. There is, however, almost no molecular level evidence that it actually occurs. My proposed research program will provide such evidence.We will create new molecular probes to test the mechanism of PITPs and lipid kinase function. This knowledge will enable a better understanding of role of PITPs in human neurological disease and potentially in the creation of drugs that stop the growth of cells. New molecules will be synthesized called “bola-lipids”, two-headed phospholipids that resemble the Native American hunting weapon. Long bola-lipids (bola-phosphatidylinositols or bola-PIs) should be good substrates for the PITP and kinase, but short ones should be bad. We will also make the bola-PIs cleavable by light or chemical reduction so that bad substrates can be turned into good ones on command. We will also incorporate specific groups (called photoaffinity labels) that can irreversibly bond with proteins that are close to the bola-PIs in a cell. Bonding to lipid kinases and/or transfer proteins will provide further evidence for the association and function of these proteins. Furthermore, we will make a fluorescent bola-lipid to allow the tracking of these compounds in artificial membranes and in living cells.