Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments

Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila

SummaryStem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts

Insight into erythrocyte phospholipid molecular flux in healthy humans and in patients with acute respiratory distress syndrome

Although the distribution of cellular membrane phospholipid composition is well characterised in human erythrocytes, in-vivo turnover and dynamic flux of phospholipids between plasma and erythrocytes in physiological and in particular during disease states are mostly unknown. Erythrocyte mass primarily consisted of lipids and phosphatidylcholine (PC) contributes to the significant proportion of phospholipid membrane composition. Esterified membrane PC can be utilised during pathological processes to generate pro and anti-inflammatory lipid mediators, which can contribute to the pathogenesis of acute respiratory distress syndrome (ARDS). In this study, utilising isotope labelling of choline and analytical methods with electrospray mass spectrometry (ESI-MS/MS), we characterised individual molecular composition and dynamic exchange of PC, sphingomyelins (SM) and lysopho-sphatidylcholines (LPC) between plasma and erythrocytes. In ARDS patients, there were significant alterations in PC molecular composition, coupled with a continuous loss of arachidonoyl-PC species over time. Infusion of methyl-D9-choline chloride resulted in enrichment of labelled choline into plasma PC and LPC via CDP-choline pathway with subsequent incorporation into erythrocyte PC. As expected, erythrocyte methyl-D9 PC enrichment is much slower than plasma. Patients had much faster and higher fractional enrichment of all PC and LPC molecules suggesting increased flux between plasma and erythrocytes. There was a particular pattern of incorporation, where the arachidonoyl-PC species achieved equilibrium with plasma rapidly and retained highest concentrations of enrichment compared to the other PC species. Increased enrichment of arachidonoyl-PC coupled with virtually no increase or depletion of its concentrations suggests the possibility of substrate donation for other cell types for the participation of eicosanoid biosynthesis during inflammatory conditions like ARDS. In summary, this study revealed an alerted pattern erythrocyte molecular phospholipid composition and flux in patients with acute respiratory distress syndrome and the pathological consequences of these changes needs further exploration.</p

Exhaled breath particles as a novel tool to study lipid composition of epithelial lining fluid from the distal lung

Abstract Background Surfactant phospholipid (PL) composition plays an important role in lung diseases. We compared the PL composition of non-invasively collected exhaled breath particles (PEx) with bronchoalveolar lavage (BAL) and induced sputum (ISP) at baseline and following endotoxin (LPS) challenges. Methods PEx and BAL were collected from ten healthy nonsmoking participants before and after segmental LPS challenge. Four weeks later, PEx and ISP were sampled in the week before and after a whole lung LPS inhalation challenge. PL composition was analysed using mass spectrometry. Results The overall PL composition of BAL, ISP and PEx was similar, with PC(32:0) and PC(34:1) representing the largest fractions in all three sample types (baseline PC(32:0) geometric mean mol%: 52.1, 56.9, and 51.7, PC(34:1) mol%: 11.7, 11.9 and 11.4, respectively). Despite this similarity, PEx PL composition was more closely related to BAL than to ISP. For most lipids comparable inter-individual differences in BAL, ISP, and PEx were found. PL composition of PEx was repeatable. The most pronounced increase following segmental LPS challenge was detected for SM(d34:1) in BAL (0.24 to 0.52 mol%) and following inhalation LPS challenge in ISP (0.45 to 0.68 mol%). An increase of SM(d34:1) following segmental LPS challenge was also detectable in PEx (0.099 to 0.103 mol%). The inhalation challenge did not change PL composition of PEx. Conclusion Our data supports the peripheral origin of PEx. The lack of PL changes in PEx after inhalation challenge might to be due to the overall weaker response of inhaled LPS which primarily affects the larger airways. Compared with BAL, which always contains lining fluid from both peripheral lung and central airways, PEx analysis might add value as a selective and non-invasive method to investigate peripheral airway PL composition. Trial registration NCT03044327, first posted 07/02/2017

Stable isotope analysis of dynamic lipidomics

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis

Mass spectrometry analysis of the phospholipase A activity of snake pre-synaptic neurotoxins in cultured neurons

Snake pre-synaptic phospholipase A(2) neurotoxins paralyse the neuromuscular junction by releasing phospholipid hydrolysis products that alter curvature and permeability of the pre-synaptic membrane. Here, we report results deriving from the first chemical analysis of the action of these neurotoxic phospholipases in neurons, made possible by the use of high sensitivity mass spectrometry. The time-course of the phospholipase A(2) activity (PLA(2)) hydrolysis of notexin, beta-bungarotoxin, taipoxin and textilotoxin acting in cultured neurons was determined. At variance from their enzymatic activities in vitro, these neurotoxins display comparable kinetics of lysophospholipid release in neurons, reconciling the large discrepancy between their in vivo toxicities and their in vitro enzymatic activities. The ratios of the lyso derivatives of phosphatidyl choline, ethanolamine and serine obtained here together with the known distribution of these phospholipids among cell membranes, suggest that most PLA(2) hydrolysis takes place on the cell surface. Although these toxins were recently shown to enter neurons, their intracellular hydrolytic action and the activation of intracellular PLA(2)s appear to contribute little, if any, to the phospholipid hydrolysis measured her

The composition of pulmonary surfactant from diving mammals

Maintaining a functional pulmonary surfactant system at depth is critical for diving mammals to ensure that inspiration is possible upon re-emergence. The lipid and protein composition of lavage extracts from three pinniped species (California sea lion, Northern elephant seal and Ringed seal) were compared to several terrestrial species. Lavage samples were purified using a NaBr discontinuous gradient. Concentrations of phospholipid classes and molecular species were measured using electrospray ionisation mass spectrometry, cholesterol was measured using high-performance liquid chromatography, surfactant protein A (SP-A) and SP-B were measured using enzyme-linked immunosorbent assays. There were small differences in phospholipid classes, with a lower level of anionic surfactant phospholipids, PG and PI, between diving and terrestrial mammals. There were no differences in PL saturation or SP-A levels between species. PC16:0/14:0, PC16:0/16:1, PC16:0/16:0, long chain PI species and the total concentrations of alkyl–acyl species of PC and PG as a ratio of diacyl species were increased in diving mammals, whereas concentrations of PC16:0/18:1, PG16:0/16:0 and PG16:0/18:1 were decreased. Cholesterol levels were very variable between species and SP-B was very low in diving mammals. These differences may explain the very poor surface activity of pinniped surfactant that we have previously described [Miller, N.J., Daniels, C.B., Schürch, S., Schoel, W.M., Orgeig, S., 2005. The surface activity of pulmonary surfactant from diving mammals

An electrospray ionization-flow tube study of H/D exchange in protonated bradykinin

An electrospray ionization-fast flow technique has. been employed to study the reactions of doubly protonated bradykinin and des-Arg(9)-bradykinin with CH3OD and ND3, respectively. Deconvolution of the experimental mass spectral data followed by simulation of the kinetic data by solution of differential equations leads to sets of apparent and site specific rate constants. On a time scale of several milliseconds, bradykinin undergoes with ND3 three fast HID exchanges and one slow exchange. Three equivalent exchanges are observed with CH3OD that are nearly 2 orders of magnitude slower than the ND3 reactions. Up to six hydrogen exchanges are observed for the reaction of des-Arg(9)-bradykinin with ND3. The more efficient exchange of des-Arg(9)- bradykinin is accompanied by formation of collisionally stabilized complexes between doubly protonated des-Arg(9)-bradykinin and ND3 at a He carrier gas pressure of about 0.2 Torr. Multiple-collision activation-collision-induced dissociation of reactant and product ions of the isotope exchange reactions was carried out in front of the sampling nose cone of the analyzer quadrupole mass filter system. The degree of deuterium incorporation into the parent doubly protonated ions and into several of the b(n)(+) and y(n)(+) ions combined with the site-specific rate constants obtained indicates that the three equivalent hydrogens exchanged in doubly protonated bradykinin are at the protonated N-terminus amine group. Complexation of doubly protonated bradykinin by ND3 is prevented by its tightly folded structure, and this in turn prevents H/D exchange of the amide hydrogens of bradykinin. The additional H/D exchanges observed in the case of doubly protonated des-Arg(9)-bradykinin are made possible by complexation of its less compact structure via hydrogen-bonded intermediates that promote H/D exchange of amide hydrogens