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

细胞包含充满水的区域，其他区域充满脂肪和脂质。生物膜是包围细胞及其内部细胞器的屏障，由在水中溶解性很差的脂类组成。然而，有时细胞需要将脂质从一个地方转移到另一个地方。研究人员已经发现了能够拾取特定脂类并将它们从一个膜位置转移到另一个膜位置的蛋白质。在过去的50年里，许多这样的蛋白质被发现并被描述，它们通常被称为“脂转移蛋白”。但它们的名字可能掩盖了我们对这些蛋白质真正功能的思考。事实上，要证明这些蛋白质在活细胞中转移脂类的能力并不是一件容易的事。我们已经制作了荧光探针来跟踪这种在哺乳动物细胞中转移维生素E的蛋白质的作用。我们还探索了携带某些磷脂的磷脂酰肌醇转移蛋白(PITPs)的功能。这些蛋白质对酵母的生存至关重要，哺乳动物形式的突变会导致各种神经疾病和发育问题。 来自酵母遗传实验的越来越多的证据表明，PITPs不仅是简单的转移蛋白，而且是与膜相关的脂激酶酶的分子助手。激酶将磷酸基团连接到分子上，一些脂质激酶产生强大的信号脂质(本质上是细胞GPS信号)，称为磷脂酰肌醇磷酸盐(PIP)。这一概念从根本上重新思考了脂转移蛋白的作用。然而，几乎没有分子水平的证据表明它确实存在。我提议的研究计划将提供这样的证据。 我们将创造新的分子探针来测试PITPs的机制和脂蛋白激酶的功能。这些知识将有助于更好地理解PITPs在人类神经疾病中的作用，并可能在创造阻止细胞生长的药物方面发挥作用。人们将合成一种名为“博拉-脂”的新分子，这是一种类似美洲原住民狩猎武器的双头磷脂。长的波拉-脂(波拉-磷脂酰肌醇或波拉-PI)应该是PITP和激酶的良好底物，但短的应该是不好的。我们还将使Bola-PI可以通过光或化学还原来分解，这样不好的底物就可以在命令下变成好的底物。我们还将加入特定的基团(称为光亲和标记)，这些基团可以与细胞中靠近Bola-PI的蛋白质不可逆地结合。与脂蛋白和/或转运蛋白的结合将为这些蛋白的结合和功能提供进一步的证据。此外，我们将制造一种荧光波拉-脂质，以允许在人造膜和活细胞中跟踪这些化合物。

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.