Biochemical Characterization of a <i>Pseudomonas aeruginosa</i> Phospholipase D

Phospholipase D is a ubiquitous protein in eukaryotes that hydrolyzes phospholipids to generate the signaling lipid phosphatidic acid (PtdOH). PldA, a <i>Pseudomonas aeruginosa</i> PLD, is a secreted protein that targets bacterial and eukaryotic cells. Here we have characterized the in vitro factors that modulate enzymatic activity of PldA, including divalent cations and phosphoinositides. We have identified several similarities between the eukaryotic-like PldA and the human PLD isoforms, as well as several properties in which the enzymes diverge. Notable differences include the substrate preference and transphosphatidylation efficiency for PldA. These findings offer new insights into potential regulatory mechanisms of PldA and its role in pathogenesis

Human Phospholipase D Activity Transiently Regulates Pyrimidine Biosynthesis in Malignant Gliomas

Cancer cells reorganize their metabolic pathways to fuel demanding rates of proliferation. Oftentimes, these metabolic phenotypes lie downstream of prominent oncogenes. The lipid signaling molecule phosphatidic acid (PtdOH), which is produced by the hydrolytic enzyme phospholipase D (PLD), has been identified as a critical regulatory molecule for oncogenic signaling in many cancers. In an effort to identify novel regulatory mechanisms for PtdOH, we screened various cancer cell lines, assessing whether treatment of cancer models with PLD inhibitors altered production of intracellular metabolites. Preliminary findings lead us to focus on how deoxyribonucleoside triphosphates (dNTPs) are altered upon PLD inhibitor treatment in gliomas. Using a combination of proteomics and small molecule intracellular metabolomics, we show herein that PtdOH acutely regulates the production of these pyrimidine metabolites through activation of CAD via mTOR signaling pathways independently of Akt. These changes are responsible for decreases in dNTP production after PLD inhibitor treatment. Our data identify a novel regulatory role for PLD activity in specific cancer types

Lipid Composition of the Viral Envelope of Three Strains of Influenza VirusNot All Viruses Are Created Equal

Although differences in the rate of virus fusion and budding from the host cell membrane have been correlated with pathogenicity, no systematic study of the contribution of differences in viral envelope composition has previously been attempted. Using rigorous virus purification, marked differences between virions and host were observed. Over 125 phospholipid species have been quantitated for three strains of influenza (HKx31-H3N2, PR8-H1N1, and VN1203-H5N1) grown in eggs. The glycerophospholipid composition of purified virions differs from that of the host or that of typical mammalian cells. Phosphatidylcholine is the major component in most mammalian cell membranes, whereas in purified virions phosphatidylethanolamine dominates. Due to its effects on membrane curvature, it is likely that the variations in its content are important to viral processing during infection. This integrated method of virion isolation with systematic analysis of glycerophospholipids provides a tool for the assessment of species-specific biomarkers of viral pathogenicity

Lipid Composition of the Viral Envelope of Three Strains of Influenza VirusNot All Viruses Are Created Equal

Although differences in the rate of virus fusion and budding from the host cell membrane have been correlated with pathogenicity, no systematic study of the contribution of differences in viral envelope composition has previously been attempted. Using rigorous virus purification, marked differences between virions and host were observed. Over 125 phospholipid species have been quantitated for three strains of influenza (HKx31-H3N2, PR8-H1N1, and VN1203-H5N1) grown in eggs. The glycerophospholipid composition of purified virions differs from that of the host or that of typical mammalian cells. Phosphatidylcholine is the major component in most mammalian cell membranes, whereas in purified virions phosphatidylethanolamine dominates. Due to its effects on membrane curvature, it is likely that the variations in its content are important to viral processing during infection. This integrated method of virion isolation with systematic analysis of glycerophospholipids provides a tool for the assessment of species-specific biomarkers of viral pathogenicity

Multi-organ Abnormalities and mTORC1 Activation in Zebrafish Model of Multiple Acyl-CoA Dehydrogenase Deficiency

<div><p>Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the <i>ETFA</i>, <i>ETFB</i>, and <i>ETFDH</i> genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant <i>dark xavier</i> (<i>dxa^vu463</i>) that has an inactivating mutation in the <i>etfa</i> gene. <i>dxa^vu463</i> recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant <i>dxa^vu463</i> zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant <i>dxa^vu463</i> zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that <i>etfa</i> gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.</p></div

Lipids, cerebroside sulfate and free cholesterol accumulations in the cytosol of type II <i>dxa^vu463</i> hepatocyte.

<p>(A) Whole mount Oil Red-O (ORO) staining of wild type and type II <i>dxa</i> at 8 dpf. Vertical line (a) indicates location of transverse section in B. (B) ORO staining in the liver sections at 8 dpf. (C) Toluidine blue, PAS and Filipin staining at 9 dpf. Wild type control livers are on the top row and <i>dxa</i> are on the bottom row. Magnified views of rectangles showing toluidine blue are in the lower left corners. The brown colored drops in Toluidine blue staining suggests cerebroside sulfate accumulation. Magnified views of rectangles showing Filipin (free cholesterol) staining are in the upper right corners. Filipin appears to accumulate in the cytosol of mutant hepatocytes. (D) TEM image at 6 dpf. Green shadows mark single representative mitochondria. Magnified views of rectangles showing cristae are on the left lower corners. (E) TEM image at 8 dpf. Nuclei are colored red and single representative mitochondria are again colored green. Dark granules in <i>dxa</i> mutants appear to represent lipid drops. Mib, midbrain; I, intestine; L, liver; bv, blood vessels; N, nucleus; cyto, cytosol; Mito, mitochondria; Lip, lipid drops. Scale bar = (A) 100 µm, (B) 50 µm, (C) 25 µm and (D, E) 2 µm.</p

Facial axons, mechanosensory hair cell and myelination defects in type II <i>dxa^vu463</i> mutant zebrafish.

<p>(A) DIC imaging of live cilia (first column) and neuromast cells (second column, asterisks), ORO stained lipids in neuromast cells (third column, asterisks). Acetylated-tubulin marks cilia (yellow arrowheads). Magnified views of cilia are shown on the upper right corner. (B) Whole mount immunofluorescence staining of acetylated-tubulin in WT (left) and <i>dxa</i> mutants (right) at 8 dpf. Yellow arrowheads indicate facial axons. Rectangle region is magnified in lower left corner. (C) anti-MBP staining in the brain (left) and spinal cord (right) in WT (top) and <i>dxa</i> mutant zebrafish (bottom) at 8 dpf. Arrows indicate myelinated axons in the spinal cord, all signal is reduced in <i>dxa</i> mutant zebrafish. (D) TEM (11,000×) image of spinal cord as indicted by arrows in C. Normal (WT) and swollen (<i>dxa</i> mutant) mitochondria are pseudocolored green, indicated by large red arrowhead). Red arrows indicate less condensed myelination layer in a <i>dxa</i> axon. Further magnified views of mitochondria are on the lower left corner. M, Mauthner axon track. Scale bars are as indicated in each panel.</p

Tissue dependent regulation of mTORC1 activation in <i>dxa^vu463</i> mutant zebrafish.

<p>Anti-phospho-S6 (left panels) and anti-phospho-4E-BP1 (right panels) antibodies were used to assess mTORC1 kinase activity. (A) WT brain (top) and <i>dxa</i> brain (bottom) at 8 dpf. Arrows indicate p-S6 and phospho-4E-BP1 positive cells in neural progenitors of the brain. P-S6 and p-4E-BP1 are also detected in the superficial pial cells of the mutant brain. (B) Sections of trunk regions in WT (top) and <i>dxa</i> (bottom) at 8 dpf. Phospho-S6 was detected in the central canal and phospho-4E-BP1 positive cells were found central canal as well as midline cells (yellow arrow) in <i>dxa</i> mutant zebrafish. Asterisks indicates central canal of hindbrain. 300 nM of rapamycin was used from 5 dpf to 8 dpf to treat <i>dxa</i> mutant zebrafish in C and D. (C) Hindbrain regions of type III mutant at 8 dpf. Phospho-S6 and phospho-4E-BP1 staining was again detected in central canal (*) and pial cell sheath (P) in both control and rapamycin treated <i>dxa</i> mutants. DAPI (blue) was used for nuclei staining. (D) Liver regions of same sections seen in (C) with marked suppression of phospho-S6 but a relative increase in phsohp-4E-BP1 levels. P, pial cell sheath; HB, hindbrain; K, kidney; L, liver. Scale bar = 100 µm.</p

Lipid accumulation and necrotic features in the <i>dxa^vu463</i> mutants with increased numbers of neural progenitor cells and dysmorphic brain.

<p>Top panels show control wild type zebrafish with bottom panels showing images from type II <i>dxa</i> mutant at 8 dpf. (A) ORO (red) showing increased lipids in the mutant brain, DAPI (bright blue) staining nuclei. (B) Toluidine blue staining in wild type and type II <i>dxa</i> with severe brain defects. Magnified view of midline region in the rectangle is shown on the upper right corner with greatly enlarged neural progenitor cells and neurons. Brown colored vesicles again suggest lipid drops containing cerebroside sulfate. (C) TEM image of VZ in <i>WT</i> (top) and type II <i>dxa</i> (bottom). Yellow pseudocolor indicates a single glia cell in <i>WT</i> and <i>dxa</i> mutants, marked increase in cell size is present. (D) Higher magnification image of neural progenitor cells. Green pseudocolor region indicates individual mitochondria. Pseudocolor with red indicates nuclei. Magnified views of normal and mutant mitochondria are shown on the left lower bottom. (E) Anti-Sox2 (red) and DAPI (blue) staining in control (top) and <i>dxa</i> zebrafish (bottom). Yellow arrows indicate Sox2 positive cells in the VZ. (F) Red channel image of (E). (G) DAPI channel of (E) showing disrupted gray matter of brain. (H) Anti-BLBP staining in wild type (top) and <i>dxa</i> (bottom). Asterisks indicate white matter region normally containing glia fibers. Region within the yellow box is further magnified to allow fine details of glial fibers to be seen. Contrast levels of control (top) and <i>dxa</i> zebrafish (bottom) were adjusted together to compare glial fibers. Scale bar = 50 µm (A, B), 10 µm (C), 2 µm (D) and 100 µm (E–H).</p

Classification of <i>dxa^vu463</i> homozygous mutants, positional cloning of <i>dxa^vu463</i> and Etfa protein expression.

<p>(A) Representative phenotypes of most severe (type I), moderate (type II) and mild (type III) <i>dxa</i> homozygous mutants at 7 dpf. Blue lines (a and b) indicate region of transverse sections in D. (B) Spectrum changes of type I, II and III mutants under different feeding conditions. Blue bars indicate the proportion of mutants under regular feeding (n = 218, 5 clutches), red bars for the proportion of mutants under extra feeding condition (n = 151, 3 clutches), p* = 0.03, p** = 0.00015. (C) Primary predicted structure of Etfa protein in wild-type and <i>dxa</i> zebrafish. Shaded codon indicates the null mutation of <i>etfa</i> in <i>dxa</i> zebrafish (GGA (Glycine) to TGA (stop)). (D) Anti-Etfa immunostaining (red) in wild-type control (upper panel, n = 9/9) and homozygous mutant (lower panel, n = 9/9) at 9 dpf. DAPI (blue) was used for nucleus staining. Arrows indicate Etfa expression in the ventricular region of the brain. Magnified midline views of yellow rectangles are in the left corners. Magnified rectangles on the trunk sections indicate neuromast hair cells. NM, neuromast; PF, pectoral fin; K, kidney; L, liver. Scale bar = 100 µm.</p