Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

Self-assembly of fibronectin into fibrillar networks underneath dipalmitoyl phosphatidylcholine monolayers: Role of lipid matrix and tensile forces

The cell-mediated assembly of fibronectin (Fn) into fibrillar matrices is a complex multistep process that is incompletely understood because of the chemical complexity of the extracellular matrix and a lack of experimental control over molecular interactions and dynamic events. We have identified conditions under which Fn assembles into extended fibrillar networks after adsorption to a dipalmitoyl phosphatidylcholine (DPPC) monolayer in contact with physiological buffer. We propose a sequential model for the Fn assembly pathway, which involves the orientation of Fn underneath the lipid monolayer by insertion into the liquid expanded (LE) phase of DPPC. Attractive interactions between these surface-anchored proteins and the liquid condensed (LC) domains leads to Fn enrichment at domain edges. Spontaneous self-assembly into fibrillar networks, however, occurs only after expansion of the DPPC monolayer from the LC phase though the LC/LE phase coexistence. Upon monolayer expansion, the domain boundaries move apart while attractive interactions among Fn molecules and between Fn and domain edges produce a tensile force on the proteins that initiates fibril assembly. The resulting fibrils have been characterized in situ by using fluorescence and light-scattering microscopy. We have found striking similarities between fibrils produced under DPPC monolayers and those found on cellular surfaces, including their assembly pathways.

Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth

Phospholipids are the major components of cell membranes and are required for cellular growth. We studied membrane phosphatidylcholine (PtdCho) biosynthesis in neuronal cells undergoing neurite outgrowth, by using PC12 cells as a model system. When neurite outgrowth was induced by exposing PC12 cells to nerve growth factor for 2 and 4 days, the amounts of [14C]choline incorporated into [14C]phosphatidylcholine per cell (i.e., per DNA) increased approximately 5- and 10-fold, respectively, as compared with control cells, reflecting increases in the rate of PtdCho biosynthesis. [14C]choline uptake was not affected. Analysis of the three major PtdCho biosynthetic enzymes showed that the activity of CDPcholine:1,2-diacylglycerol cholinephosphotransferase was increased by approximately 50% after nerve growth factor treatment, but the activities of choline kinase or choline-phosphate cytidylyltransferase were unaltered; the cholinephosphotransferase displayed a high Km value (≈1,200 μM) for diacylglycerol. Moreover, free cellular diacylglycerol levels increased by approximately 1.5- and 4-fold on the second and fourth days, respectively. These data indicate that PtdCho biosynthesis is enhanced when PC12 cells sprout neurites, and the enhancement is mediated primarily by changes in cholinephosphotransferase activity and its saturation with diacylglycerol. This suggests a novel regulatory role for diacylglycerol in membrane phospholipid biosynthesis.

Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles

The 2H,13C,15N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30°C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The 13C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.

Phosphatidylcholine Synthesis Influences the Diacylglycerol Homeostasis Required for Sec14p-dependent Golgi Function and Cell Growth

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20–40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

Evidence for an Intrinsic Toxicity of Phosphatidylcholine to Sec14p-dependent Protein Transport from the Yeast Golgi Complex

Yeast phosphatidylinositol-transfer protein (Sec14p) is essential for Golgi secretory function and cell viability. This requirement of Sec14p is relieved by genetic inactivation of the cytidine diphosphate-choline pathway for phosphatidycholine (PtdCho) biosynthesis. Standard phenotypic analyses indicate that inactivation of the phosphatidylethanolamine (PtdEtn) pathway for PtdCho biosynthesis, however, does not rescue the growth and secretory defects associated with Sec14p deficiency. We now report inhibition of choline uptake from the media reveals an efficient “bypass Sec14p” phenotype associated with PtdEtn-methylation pathway defects. We further show that the bypass Sec14p phenotype associated with PtdEtn-methylation pathway defects resembles other bypass Sec14p mutations in its dependence on phospholipase D activity. Finally, we find that increased dosage of enzymes that catalyze phospholipase D-independent turnover of PtdCho, via mechanisms that do not result in a direct production of phosphatidic acid or diacylglycerol, effect a partial rescue of sec14-1ts-associated growth defects. Taken together, these data support the idea that PtdCho is intrinsically toxic to yeast Golgi secretory function.

Mutant rat phosphatidylinositol/phosphatidylcholine transfer proteins specifically defective in phosphatidylinositol transfer: implications for the regulation of phospholipid transfer activity.

The mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PI-TPs) catalyze exchange of phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayers in vitro. We find that Ser-25, Thr-59, Pro-78, and Glu-248 make up a set of rat (r) PI-TP residues, substitution of which effected a dramatic reduction in the relative specific activity for PI transfer activity without significant effect on PC transfer activity. Thr-59 was of particular interest as it is a conserved residue in a highly conserved consensus protein kinase C phosphorylation motif in metazoan PI-TPs. Replacement of Thr-59 with Ser, Gln, Val, Ile, Asn, Asp, or Glu effectively abolished PI transfer capability but was essentially silent with respect to PC transfer activity. These findings identify rPI-TP residues that likely cooperate to form a PI head-group binding/recognition site or that lie adjacent to such a site. Finally, the selective sensitivity of the PI transfer activity of rPI-TP to alteration of Thr-59 suggests a mechanism for in vivo regulation of rPI-TP activity.