This application seeks to extend a program that has demonstrated the role of phospholipids as important ligands that regulate protein function. Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3) have been implicated in a number of fundamental cellular processes including acting as a targeting ligand for PH domain containing proteins, as an activator of signaling molecular like phospholipase D, phospholipase C (PLC), and the small G protein ARF; as a regulator of the actin cytoskeleton directing cytokinesis in Dictyostelium, and acting as a regulator of endo and exocytosis. Since these specific ligand species have important regulatory roles in processes such as membrane trafficking and cell signaling, localization of polyphosphoinositides (PIPn) within the cell is important. The first specific aim will characterize microdomains of the plasma membrane that contain PIP2 and PIP3 using genetic and biochemical approaches. It is hypothesized that specific protein complexes are involved in transporting and maintaining locally high concentrations of these (PIPn). The molecular mechanisms responsible for creation of functional compartments of bioactive lipid will be determined. The second specific aim will investigate the role of the acyl groups of phospholipids in regulating protein function. It is hypothesized that proteins like PLC delta 1 interact with the hydrophobic portions of phospholipids as well as the hydrophilic head group. Several modified forms of PIP2 such as di-C4, di-C8, and di-C16 will be assessed for activity in vitro using detergent mixed micelles with pure recombinant PLC delta 1. The specificity for acyl chains in vivo will be assessed by supplementing media with fatty acids and delivering PIP2 derivatives using polyamine carriers and looking at enzyme activity. The third specific aim will describe the allosteric effects of PS at the C2 domain of PLC delta 1. Kinetic studies will be performed in detergent mixed micelles using the soluble substrate 1,2 solution without an interface. The fourth specific aim will test the hypothesis that the acyl chains of phosphatidylserine are vital to the stimulation of PLC delta 1 via the C terminal C2 domain. Amino acid residues involved in acyl chain interactions will be identified. Because the enzymatic specificity for fatty acids is at best only moderate, the fatty acid composition of phospholipids is easily influenced by diet. Therefore, we believe that diet can influence the function of proteins and contribute to environmental phenotypes. Since many diseases that afflict man have environmental components, the DNA-protein paradigm cannot explain all human afflictions. We hypothesize that the lipids play a role in the development of human disease.
|Effective start/end date||4/1/02 → 3/31/07|
- National Heart, Lung, and Blood Institute (5 R01 HL055591-09)