1-palmitoyl-2-oleoylglycero-3-phosphoserine and dipalmitoylphosphatidylserine

HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A's cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A's PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A's lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A's secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A's ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A's refolding activities. Furthermore, using a Lysine-71-Alanine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the wild-type (WT), the mutated protein associates with lipids three times faster and dissociates 300 times faster than the WT HspA1A. These observations suggest a two-step binding model including an initial interaction of HspA1A with lipids followed by a conformational change of the HspA1A-lipid complex, which accelerates the binding reaction. Together these findings strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap and provide the basis for future interventions to inhibit HspA1A's PM-translocation in tumor cells, making them sensitive to radiation therapy.

Topics: Adenosine Triphosphate; Amino Acid Substitution; Animals; Circular Dichroism; HSP70 Heat-Shock Proteins; Liposomes; Lysine; Mice; Molecular Chaperones; Mutation; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Protein Refolding; Protein Structure, Secondary; Surface Plasmon Resonance

The interactions of the racemic mixture of articaine as well as pure (R)-articaine and pure (S)-articaine with monolayers of glycerophospholipids and brain lipids have been studied using the Langmuir monolayer technique. Articaine was added to the glycerophospholipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), 1-palmitoyl-2-oleoylphosphatidylserine (POPS) and total lipid extract from pig brain (TLPB). The amount of articaine in the monolayers was 30 mol%. The intercalation of each of the two enantiomers of articaine into a glycerophospholipid/brain lipid monolayers composed of chiral phospholipids will be diastereoisomeric in nature, hence different intercalation pattern for the two enantiomers can be expected. All the articaine species are found to intercalate into the DPPC monolayer and to increase the monolayer stability, this is most pronounced for the (R)-enantiomer. Intercalation of the articaine species into the DPPS monolayer increases the MMA and hardly affects the stability of the DPPS monolayer. In this monolayer, the articaine species intercalates into the head group region of the small and negatively charged serine head group, this is pronounced for the (R)-enantiomer. Our results indicate that by introducing an unsaturated acyl chain in the monolayer as in POPS, the monolayer discriminates between the articaine species. The (R)-enantiomer is located deep in the acyl chain region, whereas the (S)-enantiomer is found at or close to the head group. The data also might indicate that the (R)-enantiomer in the racemic mixture forms dimers in the POPS monolayer. Both articaine species as well as the racemic mixture intercalate into the monolayer of TLPB. Intercalation into this monolayer did not show any distinct difference in intercalation mode of the articaine species, probably due to camouflaging effect of large head groups like gangliosides and/or formation of lipid rafts in the monolayer. However, the (R)-enantiomer appears to intercalate better into the TLPB monolayer than the (S)-enantiomer. With proper standardization the Langmuir monolayer technique is a powerful method to discriminate between (R)- and (S)-enantiomer articaine interaction with model membranes.

Topics: Anesthetics, Local; Animals; Brain; Carticaine; Cell Membrane; Lipid Metabolism; Phosphatidylserines; Phosphorylcholine; Stereoisomerism; Swine

Non-oxidized phosphatidylserine (PS) is known to play a key role in apoptosis but there is considerable research evidence suggesting that oxidized PS also plays a role in this event, leading to the increasing interest in studying PS oxidative modifications. In this work, different PS (1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) were oxidized in vitro by hydroxyl radical, generated under Fenton reaction conditions, and the reactions were monitored by ESI-MS in negative mode. Oxidation products were then fractionated by thin layer chromatography (TLC) and characterized by tandem mass spectrometry (MS/MS). This approach allowed the identification of hydroxyl, peroxy, and keto derivatives due to oxidation of unsaturated fatty acyl chains. Oxidation products due to oxidation of serine polar head were also identified. These products, with lower molecular weight than the non-modified PS, were identified as [M - 29 - H](-) (terminal acetic acid), [M - 30 - H](-) (terminal acetamide), [M - 13 - H](-) (terminal hydroperoxyacetaldehyde), and [M - 13 - H](-) (terminal hydroxyacetaldehyde plus hydroxy fatty acyl chain). Phosphatidic acid was also formed in these conditions. These findings confirm the oxidation of the serine polar head induced by the hydroxyl radical. The identification of these modifications may be a valuable tool to evaluate phosphatidylserine alteration under physiopathologic conditions and also to help understand the biological role of phosphatidylserine oxidation in the apoptotic process and other biological functions.

Topics: Hydroxyl Radical; Oxidation-Reduction; Phosphatidylserines; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

We have earlier investigated the interaction of the antipsychotic drugs chlorpromazine(CPZ) and olanzapine(OLP) with glycerophospholipid monolayers. These experiments were carried out at high and low temperatures and showed that OLP had a more pronounced effect on the packing of the phospholipid (PL) monolayers than CPZ. At pH 7.36, where OLP consists of one positive and one neutral species. In the present work we have studied the interaction of the drugs with monolayers of PLs by the Langmuir technique at pH 6.00 and 10.00 at 37°C. The PLs were palmitoylphosphatidyl-choline(DPPC), 1-stearoyl-2-arachinodonoylphoshatidylcholine(SAPC),dipalmitoylphosphatidyl-serine(DPPS) and 1-palmitoyl-2-oleoylphosphatidylserine(POPS). OLP has a pKa around 7.4, with one neutral and one positive species at pH 6.00 and pH 10.00, respectively. CPZ has pKa value around 9.4, and is positively charged at pH 6.00 and neutral at pH 10.00. Our studies revealed that the surface area of DPPC with CPZ in the subphase did not change at pH 6.00. In contrast, OLP increased the mean molecular area(MMA) of DPPC at pH 6.00, while CPZ caused distinct increase in MMA on the monolayer packing of all the other PLs, including monolayers of DPPC at pH 10.00. OLP, increased MMA of all PLs at both pHs. Further, OLP increased MMA of DPPC (pH 10.00), SAPC (pH 10.00), DPPS (pH 6.00) and POPS (pH 6.00) at 30mN/m, the expected MMA of biological membranes. CPZ had the more pronounced effect at lift-off and gave an effect of the monolayers with negatively charged head groups in accordance our earlier experiments. However, CPZ affected the packing of the SAPC monolayer both at pH 6.00 and 10.00, and DPPC at pH 10.00. Both these PLs have neutral choline head group. Our results suggest that both drugs intercalate in the PL monolayers, and that the intercalation might involve electrostatic interaction with the head groups or hydrophobic interaction with the acyl chains of the PLs, or both. Probably the drugs intercalate to different extents depending on charge of both the drugs and the PL head groups. Our investigation may suggest that the interaction of CPZ and OLP with membrane PLs could be linked to both the psychotropic and the side effects.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Benzodiazepines; Chlorpromazine; Glycerophospholipids; Hydrogen-Ion Concentration; Olanzapine; Phosphatidylcholines; Phosphatidylserines; Psychotropic Drugs; Temperature

The typical antipsychotics chlorpromazine (CPZ) and trifluoperazine (TFP) increase the mean molecular area (mma) of acidic, but not neutral, glycerophospholipids in monolayers at pH 7.36 measured by the Langmuir technique. The atypical antipsychotic olanzapine (OLP(1)) is structurally similar to TFP. We have therefore studied the effects of OLP on glycerophospholipid monolayers and in comparison with CPZ. Olanzapine (10 microM, in subphase, pH 7.36) influenced the isotherms (surface pressure versus mma) in monolayers of the neutral dipalmitoyl phosphatidylcholine (DPPC) and the acidic dipalmitoyl phosphatidylserine (DPPS) or 1-palmitoyl-2-oleoylphosphatidylserine (POPS) in the increasing order of mma: DPPS

Topics: 1,2-Dipalmitoylphosphatidylcholine; Benzodiazepines; Glycerophospholipids; Hydrogen-Ion Concentration; Membranes, Artificial; Olanzapine; Phosphatidylserines; Promazine; Psychotropic Drugs; Surface Properties; Temperature