Regulatory mechanisms of surfactant secretion

AbstractSurfactant secretion is a critical regulated process in the metabolism of pulmonary surfactant. Presumably, because this process is vital to the survival of the organism, there are several independent pathways for stimulating secretion which work through different cell surface receptors and signaling mechanisms. In addition, there is apparent homeostatic regulation in that two components of surfactant, namely SP-A and dipalmitoylphosphatidylcholine, can inhibit secretion. Although secretion of surfactant has been studied for over two decades, there remains some important issues to be resolved. In vivo secretion can be stimulated by hyperventilation or even a single large breath. However, we do not know the biochemical mechanism for this physiologically important form of stimulation. In vitro, we know many of the proximal events in signaling, but we do not know how the lamellar bodies move within a cell or the docking mechanism at the plasma membrane. Many investigators have demonstrated that SP-A will inhibit secretion in vitro, but the mechanism is not known. Finally, there is a route of secretion of SP-A independent of lamellar bodies, but we do not know details of this pathway nor its regulation

Making heads or tails of phospholipids in mitochondria

Mitochondria are dynamic organelles whose functional integrity requires a coordinated supply of proteins and phospholipids. Defined functions of specific phospholipids, like the mitochondrial signature lipid cardiolipin, are emerging in diverse processes, ranging from protein biogenesis and energy production to membrane fusion and apoptosis. The accumulation of phospholipids within mitochondria depends on interorganellar lipid transport between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. The discovery of proteins that regulate mitochondrial membrane lipid composition and of a multiprotein complex tethering ER to mitochondrial membranes has unveiled novel mechanisms of mitochondrial membrane biogenesis

A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents

The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however, the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the APCs exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of S. cerevisiae titratableessential gene and haploid nonessential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). Fifty-eight strains out of~5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER-resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity

From protease to decarboxylase: the molecular metamorphosis of phosphatidylserine decarboxylase

Background: Phosphatidylserine decarboxylase (PSD) undergoes an autoendoproteolytic cleavage but the mechanism has not been defined. Results: PSD proenzyme processing occurs by a canonical serine protease mechanism catalyzed by a conserved aspartate-histidine-serine triad. Conclusion: PSD proenzyme executes a proteolytic reaction in cis that creates the active site of the decarboxylase. Significance: The mechanism of autoendoproteolyic processing of PSDs across phyla has been elucidated

Surfactant protein D increases fusion of Mycobacterium tuberculosis- containing phagosomes with lysosomes in human macrophages

Lung surfactant protein D (SP-D) binds to Mycobacterium tuberculosis surface lipoarabinomannan and results in bacterial agglutination, reduced uptake, and inhibition of growth in human macrophages. Here we show that SP-D limits the intracellular growth of bacilli in macrophages by increasing phagosome-lysosome fusion but not by generating a respiratory burst

ω-Carboxyl variants of 7-ketocholesteryl esters are ligands for β2-glycoprotein I and mediate antibody-dependent uptake of oxidized LDL by macrophages

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for anticardiolipin antibodies (aCL, Abs) present in patients with antiphospholipid syndrome. We recently reported that beta(2)-GPI specifically binds to oxidized LDL (oxLDL) and that the beta(2)-GPI's major ligand, oxLig-1 is 7-ketocholesteryl-9-carboxynonanoate (Kobayashi, K, E. Matsuura, Q. P. Liu, J. Furukawa, K. Kaihara, J. Inagaki, T. Atsumi, N. Sakairi, T. Yasuda, D. R. Welker, and T. Koike. 2001. A specific ligand for beta(2)-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages. J Lipid Res. 42: 697-709). In the present study, we demonstrate that omega-carboxylated 7-ketocholesteryl esters are critical for beta(2)-GPI binding. A positive ion mass spectrum of a novel ligand, designated oxLig-2, showed fragmented ions at m/z 383 and 441 in the presence of acetone, which share features of oxLig-1 and 7-ketocholesterol. In the negative ion mode, ions at m/z 627, 625, and 243 were observed. oxLig-2 was most likely 7-ketocholesteryl-12-carboxy (keto) dodecanoate. These ligands were recognized by beta(2)-GPI. Liposome binding to macrophages was significantly increased depending on the ligand's concentration, in the presence of beta(2)-GPI and an anti-beta(2)-GPI Ab. Synthesized variant, 7-ketocholesteryl-13-carboxytxidecanoate (13-COOH-7KC), also showed a significant interaction with beta(2)-GPI and a similar binding profile with macrophages. Methylation of the carboxyl function diminished all of the specific ligand interactions with beta(2)-GPI. Thus, omega-carboxyl variants of 7-ketocholesteryl esters can mediate anti-beta(2)-GPI Ab-dependent uptake of oxLDL by macrophages, and autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL

Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay

<p>Abstract</p> <p>Background</p> <p>The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite <it>Plasmodium falciparum</it>, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In <it>P. falciparum</it>, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells.</p> <p>Results</p> <p>We have employed an enzyme-coupled methylation assay to screen for potential inhibitors of PfPMT. In addition to hexadecyltrimethylammonium, previously known to inhibit PfPMT, two compounds dodecyltrimethylammonium and amodiaquine were also found to inhibit PfPMT activity <it>in vitro</it>. Interestingly, PfPMT activity was not inhibited by the amodiaquine analog, chloroquine, or other aminoquinolines, amino alcohols, or histamine methyltransferase inhibitors. Using yeast as a surrogate system we found that unlike wild-type cells, yeast mutants that rely on PfPMT for survival were sensitive to amodiaquine, and their phosphatidylcholine biosynthesis was inhibited by this compound. Furthermore NMR titration studies to characterize the interaction between amoidaquine and PfPMT demonstrated a specific and concentration dependent binding of the compound to the enzyme.</p> <p>Conclusion</p> <p>The identification of amodiaquine as an inhibitor of PfPMT <it>in vitro </it>and in yeast, and the biophysical evidence for the specific interaction of the compound with the enzyme will set the stage for the development of analogs of this drug that specifically inhibit this enzyme and possibly other PMTs.</p

Gem1 and ERMES Do Not Directly Affect Phosphatidylserine Transport from ER to Mitochondria or Mitochondrial Inheritance

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology

Isolation and Characterization of the Saccharomyces cerevisiae DPP1 Gene Encoding Diacylglycerol Pyrophosphate Phosphatase

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme fromSaccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1(diacylglycerol pyrophosphatephosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. Adpp1Δ mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1Δ mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S. cerevisiae

Smoking reduces surfactant protein D and phospholipids in patients with and without chronic obstructive pulmonary disease

<p>Abstract</p> <p>Background</p> <p>Pulmonary surfactant D (SP-D) has important regulatory functions for innate immunity and has been implicated as a biomarker for chronic obstructive pulmonary disease (COPD). We hypothesized that COPD patients would have reduced bronchoalveolar lavage (BAL) fluid SP-D levels compared to healthy smoking and non-smoking controls.</p> <p>Methods</p> <p>BAL SP-D and phospholipids were quantified and corrected for dilution in 110 subjects (65 healthy never smokers, 23 smokers with normal spirometry, and 22 smokers with COPD).</p> <p>Results</p> <p>BAL SP-D was highest in never smokers (mean 51.9 μg/mL ± 7.1 μg/mL standard error) compared to both smokers with normal spirometry (16.0 μg/mL ± 11.8 μg/mL) and subjects with COPD (19.1 μg/mL ± 12.9 μg/mL; P < 0.0001). Among smokers with COPD, BAL SP-D correlated significantly with FEV₁% predicted (R = 0.43; P < 0.05); however, the strongest predictor of BAL SP-D was smoking status. BAL SP-D levels were lowest in current smokers (12.8 μg/mL ± 11.0 μg/mL), intermediate in former smokers (25.2 μg/mL ± 14.2 μg/mL; P < 0.008), and highest in never smokers. BAL phospholipids were also lowest in current smokers (6.5 nmol ± 1.5 nmol), intermediate in former smokers (13.1 nmol ± 2.1 nmol), and highest in never smokers (14.8 nmol ± 1.1 nmol; P < 0.0001).</p> <p>Conclusions</p> <p>These data suggest that smokers, and especially current smokers, exhibit significantly reduced BAL SP-D and phospholipids compared to nonsmokers. Our findings may help better explain the mechanism that leads to the rapid progression of disease and increased incidence of infection in smokers.</p