The extraordinary chemical diversity of lipids provides the foundation for the many roles they fulfill in cellular systems, which ranges from controlling the structural and morpho¬logical properties of cell membranes to mediating crucial cell signaling events. Lipids with phosphate groups are ideally suited to affect protein functions and they have been shown to control an ever-increasing array of cell signaling events. The selective binding of proteins to lipid molecules with phosphate groups like phosphoinositides, ceramide-1-phosphate, sphingosine-1-phosphate or phosphatidic acid provides the specificity of the signaling event. Kinases and phosphatases, which upon activation alter the number of phosphate groups attached to the lipid headgroup, provide the temporal control, while the spatial control is rooted in the rich chemical functionality of the respective headgroup that gives rise to local enrichment through interactions with other membrane resident molecules. Despite the importance for lipid signaling, lipids with phosphate groups in their headgroup have not been identified as a unit with a common theme and their properties have not been investigated in a comparative manner. The experiments outlined by the PIs are designed to identify the physico¬chemical commonalities these lipid signaling molecules exhibit, while at the same time they are designed to highlight the disparities among these lipids that give rise to their selective protein binding, differences in lateral distribution and their varying subcellular localization. The PIs will investigate the extent and consequences of hydrogen bond formation between like and unlike lipid species with phosphate groups, the effect of cholesterol on the lateral distribution of these lipids species, the interaction of these lipids with arginine, lysine and histidine amino acid residues and the impact of lipid morphology on the activity of lipid modifying enzymes. The PIs will continue their partnership with the Max Planck Institute (MPI) for Colloids and Interfaces (Germany). One undergraduate student each summer will visit the MPI for a 10 week inter¬national research experience that exposes the student to a different cultural environ¬ment and at the same time allows the student to work at a premier research institution. During the academic year undergraduate students will work in the PIs research labs, which will prepare them for their summer research experience. As in the past, the PIs will strive to identify students from groups underrepresented in the sciences for these experiences.
Phosphoinositide lipids mediate an extraordinary range of cellular processes by attracting proteins to distinct cellular sites. This spatiotemporal control of protein function involves the generation of localized phosphoinositide pools by kinases and phosphatases that add or remove phosphate groups at different positions of the phosphoinositide headgroup. Using lipid model systems, this project explored the conditions that lead to the stabilization of localized phosphoinositide pools and investigated the lipid binding properties of the phosphoinositide phosphatase PTEN. The lateral distribution of phosphoinositides and their interactions with proteins are governed by the ionization state of the phosphoinositide headgroup and its ability to form intra- and intermolecular hydrogen bonds. Throughout this project, we detailed the ionic properties of phosphoinositides in the presence of magnesium or calcium cations as well as plasma membrane lipids that can form hydrogen bonds with phosphoinositides. We found that calcium ions interact more strongly with phosphoinositides than magnesium ions, however, at physiological concentrations for either cation (cellular calcium concentrations are significantly lower than those of magnesium), both cations interacted with the phosphoinositide headgroup, leading to increased ionization and changes in the lateral distribution of the lipid. For phosphatidylinositol-4,5-bisphosphate we found that the 5-phosphate interacts with the respective cation more strongly than the 4-phosphate, suggesting an inositol ring orientation where the 5-phosphate is more accessible than the 4-phosphate. It had been shown previously that cholesterol levels in cellular membranes affect phosphoinositide mediated signaling events. In line with this observation, we found that cholesterol stabilizes phosphoinositide pools. Cholesterol apparently participates via its hydroxyl group in the hydrogen bond network that is being formed between the phosphoinositide headgroups. Phosphatidylethanolamine and phosphatidylinositol are two lipids found at the inner leaflet of the plasma membrane. We found that both lipids interact with phosphoinositides via hydrogen bond formation. In particular the finding that phosphatidylinositol and phosphoinositides co-localize in domains has potentially far reaching consequences for our understanding of phosphoinositide mediated signaling events since phosphatidylinositol appears to stabilize phosphoinositide enriched pools. Phosphoinositides phosphorylated at the 3-position of the inositol ring are part of several important signaling pathways that control cell processes like cell division and programmed cell death. PTEN is a phosphatase that dephosphorylates phosphatidylinositol-3,4,5-trisphosphate, thereby antagonizing the action of phosphatidylinositol 3-kinase. Mutations in the PTEN gene have been associated with a broad range of cellular dysfunctions. We investigated the lipid binding preferences of PTEN and determined structural features of the protein in the free and membrane bound state. The PTEN protein shows only minimal penetration into the lipid bilayer and binds synergistically phosphatidylinositol-4,5-bisphosphate and an anionic lipid like phosphatidylserine or phosphatidylinositol. In the absence of phosphatidylinositol-4,5-bisphosphate, PTEN associates only transiently with lipid bilayers and diffuses during this time over long distances. In the presence of phosphatidylinositol-4,5-bisphosphate the protein associates with the membrane over longer times and diffuses shorter distances.
