The anti-apoptotic activity associated with phosphatidylinositol transfer protein alpha activates the MAPK and Akt/PKB pathway.

The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein alpha (PI-TPalpha; SPIalpha cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555-1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIalpha>wtNIH3T3>SPIbeta cells (i.e. wild type cells overexpressing PI-TPbeta). Upon incubation of SPIbeta cells with the PI-TPalpha-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-kappaB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIalpha cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIbeta cells as visualized by confocal laser scanning microscopy.

Photolon, a chlorin e6 derivative, triggers ROS production and light-dependent cell death via necrosis.

Photolon is a photosensitiser with demonstrated potential as an anti-tumour agent. In this study, an in vitro investigation was performed to determine the mechanism of Photolon-induced cell death. Cell killing was observed in a light-dependent manner and light-activated Photolon resulted in a significant production of reactive oxygen species (ROS), which could be blocked by type I ROS scavengers. Inhibition of ROS production using Trolox prevented Photolon-induced cell death. Light-activated Photolon caused no increase in caspase-3/7 activity, but a rapid increase in lactate dehydrogenase (LDH) release suggesting a loss of membrane integrity and subsequent cell death by necrosis. We conclude that the mechanism of Photolon-induced cell death involves the induction of ROS via a type I mechanism, which is ultimately responsible for cell killing by necrosis.

The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein beta.

Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein beta (PI-TPbeta, SPIbeta cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPIbetaS262A cells overexpressing a mutant PI-TPbeta that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable with wtNIH3T3 cells. This suggests that the phosphorylation of Ser-262 in PI-TPbeta is involved in the regulation of apoptosis. Conditioned medium (CM) from wtNIH3T3 cells contains bioactive factors, presumably arachidonic acid metabolites [H. Bunte, et al., 2006; M. Schenning, et al., 2004] that are able to protect SPIbeta cells against UV-induced apoptosis. CM from SPIbeta cells lacks this protective activity. However, after heat denaturation CM from SPIbeta cells regains a protective activity comparable with that of wtNIH3T3 cells. This indicates that CM from SPIbeta cells contains an antagonistic factor interfering with the anti-apoptotic activity present. SPIbetaS262A cells do not produce the antagonist suggesting that phosphorylation of Ser-262 is required. Moreover, in line with the apparent lack of anti-apoptotic activity, CM from SPIbeta cells does not induce the expression of COX-2 or the activation of p42/p44 MAP kinase in SPIbeta cells. In contrast, CM from wtNIH3T3 and SPIbetaS262A cells or heat-treated CM from SPIbeta cells does induce these anti-apoptotic markers. Since we have previously shown that some of the arachidonic acid metabolites present in CM from wtNIH3T3 cells are prostaglandin (PG) E(2) and PGF(2alpha), we investigated the effect of these PGs on cell survival. Although PGE(2) and PGF(2alpha) were found to protect wtNIH3T3 and SPIbetaS262A cells against UV-induced apoptosis, these PGs failed to rescue SPIbeta cells. The fact that the concentrations of PGE(2) and PGF(2alpha) in the CM from SPIbeta cells and wtNIH3T3 cells were found to be comparable suggests that the failure of these PGs to protect SPIbeta cells could render these cells more apoptosis sensitive. Concomitantly, upon incubation with PGE(2) and PGF(2alpha), an increased expression of COX-2 and activation of p42/p44 MAP kinase were observed in wtNIH3T3 and SPIbetaS262A cells but not in SPIbeta cells. Hence, it appears that specific mechanisms of cell survival are impaired in SPIbeta cells.

Phospholipid transfer proteins in perspective.

Since their discovery and subsequent purification from mammalian tissues more than 30 years ago an impressive number of studies have been carried out to characterize and elucidate the biological functions of phosphatidylcholine transfer protein (PC-TP), phosphatidylinositol transfer protein (PI-TP) and non-specific lipid transfer protein, more commonly known as sterol carrier protein 2 (SCP-2). Here I will present information to show that these soluble, low-molecular weight proteins constitute domain structures in StArR-related lipid transfer (START) proteins (i.e. PC-TP), in retinal degeneration protein, type B (RdgB)-related PI-TPs (e.g. Dm RdgB, Nir2, Nir3) and in peroxisomal beta-oxidation enzyme-related SCP-2 (i.e. 3-oxoacyl-CoA thiolase, also denoted as SCP-X and the 80-kDa D-bifunctional protein). Further I will summarize the most recent studies pertaining to the physiological function of these soluble phospholipid transfer proteins in metazoa.

Production of reactive oxygen species in mitochondria of HeLa cells under oxidative stress.

Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed ("dark") accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.

A phosphatidylinositol transfer protein alpha-dependent survival factor protects cultured primary neurons against serum deprivation-induced cell death.

Selective neuronal loss is a prominent feature in both acute and chronic neurological disorders. Recently, a link between neurodegeneration and a deficiency in the lipid transport protein phosphatidylinositol transfer protein alpha (PI-TPalpha) has been demonstrated. In this context it may be of importance that fibroblasts overexpressing PI-TPalpha are known to produce and secrete bioactive survival factors that protect fibroblasts against UV-induced apoptosis. In the present study it was investigated whether the conditioned medium of cells overexpressing PI-TPalpha (CMalpha) has neuroprotective effects on primary neurons in culture. We show that CMalpha is capable of protecting primary, spinal cord-derived motor neurons from serum deprivation-induced cell death. Since the conditioned medium of wild-type cells was much less effective, we infer that the neuroprotective effect of CMalpha is linked (in part) to the PI-TPalpha-dependent production of arachidonic acid metabolites. The neuroprotective activity of CMalpha is partly inhibited by suramin, a broad-spectrum antagonist of G-protein coupled receptors. Western blot analysis shows that brain cortex and spinal cord express relatively high levels of PI-TPalpha, suggesting that the survival factor may be produced in neuronal tissue. We propose that the bioactive survival factor is implicated in neuronal survival. If so, PI-TPalpha could be a promising target to be evaluated in studies on the prevention and treatment of neurological disorders.

Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore.

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-gamma) or TNF-alpha could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membrane-impermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-gamma- and TNF-alpha-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M(r) 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthase-beta, alpha-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.

"Wages of fear": transient threefold decrease in intracellular ATP level imposes apoptosis.

In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10-35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H(2)O(2)-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular "ATP-meter(s)" generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).

Overexpression of phosphatidylinositol transfer protein beta in NIH3T3 cells has a stimulatory effect on sphingomyelin synthesis and apoptosis.

Phosphatidylinositol transfer proteins (PI-TPs) consist of two isoforms (PI-TPalpha and PI-TPbeta), which differ in phospholipid transfer properties and intracellular localization. Both PI-TP isoforms are substrates for protein kinase C and contain a minor phosphorylation site (Ser166 in PI-TPalpha; Ser165 in PI-TPbeta). Only PI-TPbeta contains a major phosphorylation site at Ser262, which must be phosphorylated for PI-TPbeta to be associated with the Golgi. The PI-TP isoforms are completely conserved between mammals. Although their function is still not clear, their importance follows from knock-out studies, showing that mice lacking PI-TPalpha die soon after birth and that embryonic stems cells lacking PI-TPbeta cannot be generated [Mol. Biol. Cell 13 (2002) 739]. We determined the levels of the PI-TP isoforms in various mouse tissues by immunoblotting. PI-TPalpha is present in all tissues investigated, with highest levels in brain (167 ng/100 microg total protein). The levels of PI-TPbeta are 50-100 times lower than those of PI-TPalpha, with relatively high levels found in liver and brain (1.2 and 1.8 ng/100 microg of total protein, respectively). In contrast to NIH3T3 cells overexpressing PI-TPalpha, cells overexpressing PI-TPbeta (SPIbeta cells) were able to maintain steady-state levels of sphingomyelin in plasma membrane under conditions where this lipid is degraded by exogenous sphingomyelinase. This process of rapid sphingomyelin replenishment is dependent on PI-TPbeta being associated with the Golgi as cells overexpressing a mutant PI-TPbeta in which the major phosphorylation site is replaced (PI-TPbeta(S262A) behave as wild-type NIH3T3 cells. Since the SPIbeta cells display a decreased growth rate (35 h as compared to 21 h for wtNIH3T3 cells), we have investigated the sensitivity of these cells towards UV-induced apoptosis. We have found that the SPIbeta cells, but not the cells overexpressing PI-TPbeta(S262A), are very sensitive. We are currently investigating whether a relationship exists between PI-TPbeta being involved in maintaining plasma membrane sphingomyelin levels and the enhanced sensitivity towards apoptosis.

Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor.

Mouse fibroblast cells overexpressing phosphatidylinositol transfer protein alpha [PI-TPalpha; sense PI-TPalpha (SPIalpha) cells] show a significantly increased rate of proliferation and an extreme resistance toward ultraviolet- or tumor necrosis factor-alpha-induced apoptosis. The conditioned medium (CM) from SPIalpha cells or the neutral lipid extract from CM stimulated the proliferation of quiescent wild-type NIH3T3 cells. CM was also highly effective in increasing resistance toward induced apoptosis in both wild-type cells and the highly apoptosis-sensitive SPIbeta cells (i.e., wild-type cells overexpressing PI-TPbeta). CM from SPIalpha cells grown in the presence of NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, expressed a diminished mitogenic and antiapoptotic activity. This strongly suggests that at least one of the bioactive factor(s) is an eicosanoid. In accordance, SPIalpha cells express enhanced levels of COX-1 and COX-2. The antiapoptotic activity of CM from SPIalpha cells tested on SPIbeta cells was inhibited by approximately 50% by pertussis toxin and suramin as well as by SR141716A, a specific antagonist of the cannabinoid 1 receptor. These inhibitors had virtually no effect on the COX-2-independent antiapoptotic activity of CM from SPIalpha cells. The latter results imply that PI-TPalpha mediates the production of a COX-2-dependent eicosanoid that activates a G-protein-coupled receptor, most probably a cannabinoid 1-like receptor.

Regulation of sterol carrier protein gene expression by the forkhead transcription factor FOXO3a.

The SCP gene encodes two proteins, sterol carrier protein X (SCPx) and SCP2, that are independently regulated by separate promoters. SCPx has been shown to be the thiolase involved in the breakdown of branched-chain fatty acids and in the biosynthesis of bile acids. The in vivo function of SCP2 however remains to be established. The transcriptional regulation of SCPx and SCP2 is unclear, but their promoter regions contain several putative regulatory domains. We show here that both SCPx and SCP2 are upregulated by the daf-16-like Forkhead transcription factor FOXO3a (also known as FKHRL1) on the level of promoter activity. It was recently described that Forkheads regulate protection against (oxidative) stress in both Caenorhabditis elegans and mammalian cells. We looked into a role for SCP2 in the cellular defense against oxidative damage and found that a fluorescent fatty acid analog bound to SCP2 is protected against H2O2/Cu2+-induced oxidative damage. We propose a model for the way in which SCP2 could protect fatty acids from peroxidation.

Photodynamic treatment and H2O2-induced oxidative stress result in different patterns of cellular protein oxidation.

Photodynamic treatment (PDT) is an emerging therapeutic procedure for the management of cancer, based on the use of photosensitizers, compounds that generate highly reactive oxygen species (ROS) on irradiation with visible light. The ROS generated may oxidize a variety of biomolecules within the cell, loaded with a photosensitizer. The high reactivity of these ROS restricts their radius of action to 5-20 nm from the site of their generation. We studied oxidation of intracellular proteins during PDT using the ROS-sensitive probe acetyl-tyramine-fluorescein (acetylTyr-Fluo). This probe labels cellular proteins, which become oxidized at tyrosine residues under the conditions of oxidative stress in a reaction similar to dityrosine formation. The fluorescein-labeled proteins can be visualized after gel electrophoresis and subsequent Western blotting using the antibody against fluorescein. We found that PDT of rat or human fibroblasts, loaded with the photosensitizer Hypocrellin A, resulted in labeling of a set of intracellular proteins that was different from that observed on treatment of the cells with H2O2. This difference in labeling patterns was confirmed by 2D electrophoresis, showing that a limited, yet distinctly different, set of proteins is oxidized under either condition of oxidative stress. By matching the Western blot with the silver-stained protein map, we infer that alpha-tubulin and beta-tubulin are targets of PDT-induced protein oxidation. H2O2 treatment resulted in labeling of endoplasmic reticulum proteins. Under conditions in which the extent of protein oxidation was comparable, PDT caused massive apoptosis, whereas H2O2 treatment had no effect on cell survival. This suggests that the oxidative stress generated by PDT with Hypocrellin A activates apoptotic pathways, which are insensitive to H2O2 treatment. We hypothesize that the pattern of protein oxidation observed with Hypocrellin A reflects the intracellular localization of the photosensitizer. The application of acetylTyr-Fluo may be useful for characterizing protein targets of oxidation by PDT with various photosensitizers.

Oxidation of ER resident proteins upon oxidative stress: effects of altering cellular redox/antioxidant status and implications for protein maturation.

Previous work showed that from all cellular proteins, the endoplasmic reticulum (ER) resident proteins are most sensitive to oxidative stress [hydrogen peroxide (H(2)O(2))], as determined using the oxidation-sensitive, membrane-permeable, acetylTyrFluo probe. Because of the importance of these proteins in proper cellular functioning, we studied (a) whether modifying the cellular redox state/antioxidant status alters the susceptibility of those proteins toward H(2)O(2) oxidative stress and (b) whether H(2)O(2) affects ER function with regard to protein folding. The cellular redox and/or antioxidative capacity was modified in several ways. Lowering the capacity increased H(2)O(2)-induced protein oxidation, and increasing the capacity lowered H(2)O(2)-induced protein oxidation. The effect of H(2)O(2) on ER-related protein maturation was investigated, using the maturation of the low-density lipoprotein receptor as a model. Its maturation was not affected at low concentrations of H(2)O(2) (< or = 400 micro M), which do result in oxidation of ER resident proteins. Maturation was slowed down or reversibly inhibited at higher concentrations of H(2)O(2) (1.5-2.0 mM). These results might be caused by several events, including oxidation of the low-density lipoprotein receptor itself or ER resident proteins resulting in decreased folding (capacity). Alternatively, oxidation of cytosolic proteins involved in ER Golgi transport might attenuate transport and maturation. Clearly, the mechanism(s) responsible for the impairment of maturation need further investigation.

The structure of phosphatidylinositol transfer protein alpha reveals sites for phospholipid binding and membrane association with major implications for its function.

Elucidation of the three-dimensional structure of phosphatidylinositol transfer protein alpha (PI-TPalpha) void of phospholipid revealed a site of membrane association connected to a channel for phospholipid binding. Near the top of the channel specific binding sites for the phosphorylcholine and phosphorylinositol head groups were identified. The structure of this open form suggests a mechanism by which PI-TPalpha preferentially binds PI from a membrane interface. Modeling predicts that upon association of PI-TPalpha with the membrane the inositol moiety of bound PI is accessible from the medium. Upon release from the membrane PI-TPalpha adopts a closed structure with the phospholipid bound fully encapsulated. This structure provides new insights as to how PI-TPalpha may play a role in PI metabolism.

Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress.

Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and we conclude that this model also extends to mammalian systems.

Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide.

The membrane-permeable fluorescein-labelled tyramine conjugate (acetylTyrFluo) was used to identify the proteins of normal human dermal fibroblasts most susceptible to oxidation by hydrogen peroxide [Van der Vlies, Wirtz and Pap (2001) Biochemistry 40, 7783-7788]. By exposing the cells to H(2)O(2) (0.1 mM for 10 min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [(35)S]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the (35)S-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the (35)S-labelled proteins and identified by comparison with our mastermap of proteins. Protein disulphide isomerase (PDI), IgG-binding protein (BiP), calnexin, endoplasmin and glucose-regulated protein 58 (endoplasmic reticulum protein 57/GRP58) were identified as targets of oxidation. All these proteins reside in the endoplasmic reticulum and are part of the protein folding machinery. In agreement, confocal laser scanning microscopy showed co-localization of TyrFluo-labelled proteins and the KDEL receptor ERD-2, a marker for the endoplasmic reticulum.

Structure of apo-phosphatidylinositol transfer protein alpha provides insight into membrane association.

Phosphatidylinositol transfer protein alpha (PITP alpha) is a ubiquitous and highly conserved protein in multicellular eukaryotes that catalyzes the exchange of phospholipids between membranes in vitro and participates in cellular phospholipid metabolism, signal transduction and vesicular trafficking in vivo. Here we report the three-dimensional crystal structure of a phospholipid-free mouse PITP alpha at 2.0 A resolution. The structure reveals an open conformation characterized by a channel running through the protein. The channel is created by opening the phospholipid-binding cavity on one side by displacement of the C-terminal region and a hydrophobic lipid exchange loop, and on the other side by flattening of the central beta-sheet. The relaxed conformation is stabilized at the proposed membrane association site by hydrophobic interactions with a crystallographically related molecule, creating an intimate dimer. The observed open conformer is consistent with a membrane-bound state of PITP and suggests a mechanism for membrane anchoring and the presentation of phosphatidylinositol to kinases and phospholipases after its extraction from the membrane. Coordinates have been deposited in the Protein Data Bank (accession No. 1KCM).

The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

Activated neutrophils oxidize extracellular proteins of endothelial cells in culture: effect of nitric oxide donors.

Protein oxidation of human umbilical-vein endothelial cells (HUVEC) in culture was examined under various conditions of oxidative stress. Extracellular protein (ECP) oxidation was assessed by determining dityrosine bond formation, which is indicated by the covalent coupling of the membrane-impermeable tyramine-fluorescein conjugate (TyrFluo) to HUVEC proteins. The acetylated membrane-permeable form of TyrFluo (acetylTyrFluo) was used for the determination of intracellular protein (ICP) oxidation. Oxidative stress was induced by exposing the HUVEC to PMA-activated human neutrophils, to a horseradish peroxidase/hydrogen peroxide (HRP/H(2)O(2)) system or to H(2)O(2) alone. Coupling of the probes was determined by confocal laser scanning microscopy and by Western blotting using anti-fluorescein antibody. Diethylamine nitric oxide (DEANO) was used to determine the effect of NO on the tyrosyl radical formation in proteins. The oxidative burst generated by activated neutrophils for 15 min, resulted in inducing dityrosine formation in ECP of HUVEC. Similar results were obtained with HRP/H(2)O(2), but H(2)O(2) alone did not have any effect on ECP. In the presence of DEANO (0.1 mM or higher), ECP oxidation was almost completely inhibited. This indicates that NO may protect endothelial cells against protein oxidation by activated neutrophils under pro-inflammatory conditions. Activated neutrophils did not oxidize ICP of HUVEC, which strongly suggests that the effect of the oxidative burst was restricted to the proteins exposed to the medium.