Our interests in bioorganic chemistry include activity in organic synthesis as well as protein and lipid biochemistry, structural biology, and analytical biochemistry.
Strong collaborations with active groups in the Canada, the US, and Europe mean that we are directly involved with aspects of vitamin E (tocopherol) biokinetics, intracellular location, the mechanism of action of the tocopherol transfer protein (TTP), and other lipid transfer proteins, as well as proteins that interact with phospholipid membranes.
We have prepared 22 different fluorescent forms of tocopherol some of which have been used to follow intracellular and tissue location of this vitamin. Our newest labels are now being used in 3D-imaging of mice brains to explore the role of vitamin E in neuron and brain health.
We have also designed and synthesized compounds that disrupt the normal oxidative metabolism of tocopherols and may aid in the understanding of the fate of the different forms of vitamin E and the potential of tocotrienols as therapeutics for human health.
Lipid Transfer and Binding Proteins
Every hydrophobic ligand would have trouble moving about the aqueous portion of cells were it not for specific transfer proteins. Transfer proteins exist for retinoids, sterols, fatty acids, phospholipids, cholesterol, vitamin D and, of course, vitamin E (tocopherols). Each ligand has particular biochemistry to which the transfer protein must be attuned.
For instance, the sterol binding proteins (SBP) and ceramide transfer proteins (CERT) are coordinated so that the biosynthesis of both cholesterol and ceramides are controlled together. Since tocopherols are vitamins, there is no necessity for controlling biosynthesis in mammalian tissue, but we, and our collaborators, have shown that the tocopherol transfer protein (TTP) has a critical role to play in transporting tocopherol to particular membranes involved in vesicular traffic (i.e. VLDL secretion from liver cells) or to those membranes that are prone to lipid peroxidation.
We are looking at the features that control the rates of tocopherol delivery to membranes by TTP. To this end we have developed FRET assays using our fluorescent tocopherol derivative NBD-alpha-tocopherol. By varying the phospholipid composition of membrane acceptor, as well as making mutations in the transfer protein, we (along with Dr. Danny Manor
, at Case Western Reserve University) have begun to decipher those features necessary for membrane recognition by TTP, and phospholipids that effect efficient transfer. Our most recent work focuses on the role of tocopherol in neurons and astrocytes. Vitamin E deficiency can lead to critical pathologies of the nervous system, especially cerebellar ataxias.
Other proteins of interest include the phosphatidylinositol binding proteins (PITPs) Sec14p (from yeast) and the mammalian orthologues PITPalpha and PITPbeta. Using a fluorescent form of phosphatidylcholine (PC) we are able to show the ability of these proteins to transfer PC to membranes and how the rate of this transfer is affected by membrane curvature and composition. Particularly important are the so-called addressing-lipids called phosphatidylinositol phosphates (PIPs). The oxysterol binding protein (OSBP) is particularly sensitive to the presence of membrane PIPs and we have shown that PI(4)P is extracted from the membrane by OSBP. Current work is exploring the role of sterols in modifying membrane and PIP recognition.
We base our protein experiments on two major techniques. One, mentioned above, is fluorescence energy resonance transfer (FRET), and the other is called Dual Polarization Interferometry (DPI) using Biolin Scientific’s AnaLight200. In brief, this is a surface adsorption technique that allows us to adsorb phospholipid vesicles onto a sensor chip creating immobilized bilayers. We can then measure the thickness, density, and mass of this layer. Stable phospholipid layers can also have protein adsorbed to them. From similar measurements of mass and thickness, we can calculate affinities of proteins for particular phospholipid membranes. All of this work lends support to the proposed mechanism(s) of these protein in vivo.
Synthesis of New Forms of Tocopherols and Tocotrienols
We remain active in the organic synthesis of tocols and other hydrophobic compounds as tools for biochemical research. This has included a number of efforts such as:
• photoaffinity labels based on alpha-tocopherol, and other immobilized affinity ligands for retrieving novel tocol binding proteins from tissues
• fluorescent tocols
• isotopically substituted tocols (2H and 14C)
• inhibitors of tocol metabolism