Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins.

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Lipid oxidation products, including lysophosphatidylcholine (lysoPC) inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo, in part by activating phosphatidylinositol 3-kinase (PI3K), which increases the externalization of canonical transient receptor potential 6 (TRPC6) channels and the subsequent increase in intracellular calcium. Inhibition of PI3K is a potential method to decrease TRPC6 activation and restore migration, but PI3K is involved in multiple intracellular signaling pathways and has multiple downstream effectors. The goal of this study is to identify the specific p110 catalytic subunit isoforms responsible for lysoPC-induced TRPC6 externalization to identify a target for intervention while minimizing impact on alternative signaling pathways. Down-regulation of the p110α and p110δ isoforms, but not the p110β or p110γ isoforms, with small interfering RNA significantly decreased phosphatidylinositol (3,4,5)-trisphosphate production and TRPC6 externalization, and significantly improved EC migration in the presence of lysoPC. These results identify an additional role of p110α in EC and reveal for the first time a specific role of p110δ in EC, providing a foundation for subsequent in vivo studies to investigate the impact of p110 isoform inhibition on arterial healing after injury.

Objective: Previous studies showed the benefit of canonical transient receptor potential 6 (TRPC6) channel deficiency in promoting endothelial healing of arterial injuries in hypercholesterolemic animals. Long-term studies utilizing a carotid wire-injury model were undertaken in wild-type (WT) and TRPC6 −/− mice to determine the effects of TRPC6 on phenotypic modulation of vascular smooth muscle cells (SMC) and neointimal hyperplasia. We hypothesized that TRPC6 was essential in the maintenance or reexpression of a differentiated SMC phenotype and minimized luminal stenosis following arterial injury. Methods: The common carotid arteries (CCA) of WT and TRPC6 −/− mice were evaluated at baseline and 4 weeks after wire injury. At baseline, CCA of TRPC6 −/− mice had reduced staining of MYH11 and SM22, fewer elastin lamina, luminal dilation, and wall thinning. After carotid wire injury, TRPC6 −/− mice developed significantly more pronounced luminal stenosis compared with WT mice. Injured TRPC6 −/− CCA demonstrated increased medial/intimal cell number and active cell proliferation when compared with WT CCA. Immunohistochemistry suggested that expression of contractile biomarkers in medial SMC were essentially at baseline levels in WT CCA at 28 days after wire injury. By contrast, at 28 days after injury medial SMC from TRPC6 −/− CCA showed a significant decrease in the expression of contractile biomarkers relative to baseline levels. To assess the role of TRPC6 in systemic arterial SMC phenotype modulation, SMC were harvested from thoracic aortae of WT and TRPC6 −/− mice and were characterized. TRPC6 −/− SMC showed enhanced proliferation and migration in response to serum stimulation. Expression of contractile phenotype biomarkers, MYH11 and SM22, was attenuated in TRPC6 −/− SMC. siRNA-mediated TRPC6 deficiency inhibited contractile biomarker expression in a mouse SMC line. Conclusions: These results suggest that TRPC6 contributes to the restoration or maintenance of arterial SMC contractile phenotype following injury. Understanding the role of TRPC6 in phenotypic modulation may lead to mechanism-based therapies for attenuation of IH.

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A 2 (PLA 2 ), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA 2 in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA 2 enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA 2 involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA 2 α, cPLA 2 γ, iPLA 2 β, or iPLA 2 γ. Downregulation of the β isoform of iPLA 2 blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.

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Protein–lipid interactions govern the structure and function of lipoprotein particles, which transport neutral lipids and other hydrophobic cargo through the blood stream. Apolipoproteins cover the surface of lipoprotein particles, including low-density (LDL) and high-density (HDL) lipoproteins, and determine their function. Previous work has focused on small peptides derived from these apolipoproteins or used such artificial lipid systems as Langmuir monolayers or the lipid disc assay to determine how apolipoproteins interact with the neutral lipid interface. Here, we focus on a recurring protein domain found in many neutral lipid-binding proteins, the amphipathic α-helix bundle. We use liquid droplet tensiometry to investigate protein–lipid interactions on an oil droplet, which mimics the real lipoprotein interface. The N-terminus of apoE 3 and full-length apoLp-III serve as model proteins. We find that each protein interacts with lipid monolayers at the oil–aqueous interface in unique ways. For the first time, we show that helix bundle unfolding is critical for proper protein insertion into the lipid monolayer at the oil–aqueous interface and that specific membrane lipids promote the rebinding of protein upon fluctuation in droplet size. These results shed new light on how amphipathic apolipoprotein α-helix bundles interact with neutral lipid particles.

Using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) as a model amphitropic protein, we are investigating how membrane structure and composition affect protein-membrane interactions. Previous work showed that BtPI-PLC specifically binds to phosphatidylcholine (PC)-rich membranes and preferentially interacts with unilamellar vesicles with high curvature. In this work, we monitored single fluorescently labeled BtPI-PLC proteins as they cycled on and off surface-tethered phosphatidylglycerol (PG)/PC small unilamellar vesicles (SUVs) using total internal reflection fluorescence (TIRF) microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of protein-membrane interactions as well as the curvature dependence. BtPI-PLC residence times on SUVs average 300 ms, similar to published residence times (300-400 ms) for other amphitropic proteins that transiently interact with cell surfaces. The kinetics of PI-PLC/membrane interactions is well explained by a simple two state binding model with dissociation and association rate constants averaging 3 s À1 and 0.6 mM À1 s À1 respectively. In addition fluorescence correlation spectroscopy (FCS) measurements indicate that introducing lipid packing defects PG/PC SUVs by incorporating low mole percentages of dioleoylglycerol (DOG) enhances BtPI-PLC binding to SUVs. By combining these single molecule fluorescence results with previous biophysical measurements and molecular dynamics simulations, we have developed a quantitative model showing how the bacterial virulence factor Bt-PI-PLC interacts with cell membranes in molecular detail. 3629-Pos Board B357 Interplay of Membrane Lipids Differentially Affects Lipid Binding of Phosphatidic Acid Effectors