A multitude of hydrophobic lipids are present within the cell. Their distribution between the aqueous milieu of the cytosol and amphipathic membranes, and between different organellar membranes, is remarkably variant. In addition to their direct involvement as substrates and intermediates for energy storage and utilization, lipids play key roles in determining membrane physical-chemical properties as well as in regulating gene expression and intracellular signal transduction pathways. Intracellular lipid-binding proteins (LBPs) are thought to participate in establishing and maintaining the spatial and compositional variabilities in membrane structure and in regulating the activities of lipid involvement in metabolism, cell signaling, and gene regulation. Discerning their precise functions at the molecular level, however, has posed challenges. While in vitro studies establish binding affinities and specificities, and structural determinants of lipid binding and LBP-membrane interactions, intracellular activities and physiological functions are less amenable to reductionist methodologies. The LBP field has therefore used a combination of biochemical, biophysical, molecular genetic, and physiological approaches to undertake analysis of the specific functions of these proteins. This talk will focus on studies of two types of LBPs, the large multigene family of Fatty Acid Binding Proteins (FABPs) which present with distinct and overlapping tissue distributions, and the cholesterol-binding protein Niemann Pick C2 (NPC2), expressed in the endolysosomal compartment in all tissues. Initially named according to the first tissue in which they were identified, it is now known that the mammalian FABP family is comprised of a dozen separate gene products with unique and intersecting tissue expression patterns. All bind long chain fatty acids, thus we asked why so many different proteins had evolved, and why multiple FABPs may be expressed in a single cell type. We demonstrated that, despite similar tertiary structures and equilibrium binding properties, different members of the FABP family have dramatically different mechanisms of ligand transport to membranes and have identified the protein structural domains responsible for these differences, and the membrane properties that promote FABP-membrane interactions. Studies of mice null for different FABPs, particularly those that are co-expressed in the small intestine, have further revealed the unique functions of these proteins in fatty acid and endocannabinoid uptake and transport. Indeed, the effects of FABPs are evident not only in their tissues of origin, but also in their regulation of peripheral tissues and systemic metabolism. The cholesterol-binding NPC2 protein, unlike the FABPs, is expressed in all tissues. We demonstrated its role in intracellular cholesterol transport, and its functional and specific interaction with the unique lysosomal phospholipid lysobisphosphatidic acid (LBPA; also known as bismonoacylglycerol phosphate or BMP). We have further shown that enrichment of NPC1-deficient cells with LBPA via its metabolic precursor phosphatidylglycerol, leads to sterol redistribution and clearance via exosome biogenesis and the stimulation of autophagy. Thus, cellular cholesterol reduction using LBPA enrichment is being explored as a new therapeutic approach to the neurodegenerative storage disorder, Niemann Pick C.

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