Membrane Phospholipid Catabolism Research Articles

The binding of autotaxin to integrins mediates hyperhomocysteinemia-potentiated platelet activation and thrombosis in mice and humans

AbstractHyperhomocysteinemia (HHcy) is associated with an exaggerated platelet thrombotic response at sites of vascular injury. In this study, human medical examination showed that elevated human plasma Hcy levels correlated positively with enhanced blood coagulation and platelet activity, suggesting that humans with HHcy are more prone to thrombus formation at the sites of vascular injury. Accordingly, we observed accelerated platelet activation, primary hemostasis, and thrombus formation in apolipoprotein E-deficient (ApoE−/−) mice with acute or chronic HHcy. Upon homocysteine (Hcy) administration in C57BL/6J mice, platelet aggregation, spreading and clot retraction were markedly induced. More important, Hcy increased the affinity of platelet integrin αIIbβ3 with ligands and enhanced integrin outside-in signaling by promoting membrane phosphatidylserine exposure in vitro. Mechanistically, lipidomics analysis showed that lysophosphatidylcholines were the primary metabolites leading to clustering of HHcy-stimulated platelets. Cytosolic phospholipase A2 (cPLA2) activity and autotaxin (ATX, a secreted lysophospholipase D) secretion were upregulated by Hcy, leading to membrane phospholipid hydrolysis and PS exposure. Moreover, secreted ATX directly interacted with integrin β3. Inhibitors of cPLA2 and ATX activity blocked integrin αIIbβ3 outside-in signaling and thrombosis in HHcy ApoE−/− mice. In this study, we identified a novel mechanism by which HHcy promotes platelet membrane phospholipid catabolism and extracellular ATX secretion to activate integrin outside-in signaling, consequently exacerbating thrombosis and the results revealed an innovative approach to treating HHcy-related thrombotic diseases.

Neurometabolic alterations in bipolar disorder with anxiety symptoms: A proton magnetic resonance spectroscopy study of the prefrontal whiter matter

To identify the pathophysiological mechanism of bipolar disorder (BD) patients with anxiety symptoms, we analyzed the differences of brain biochemical metabolism in BD patients with and without anxiety symptoms. We collected 39 BD patients who had been untreated with drugs in one month and were divided into the anxiety symptoms group (20 cases) and non-anxiety symptoms group (19 cases) according to whether they had anxiety symptoms. We used proton magnetic resonance spectroscopy (1H-MRS) to detect the biochemical metabolite ratios of the prefrontal whiter matter (PWM) in all patients. The right PWM mI/Cr ratios in BD patients with anxiety symptoms were higher than those in BD patients without anxiety symptoms and the Cho/Cr ratios in the left PWM were negatively correlated with age and age of onset in BD patients with anxiety symptoms. These findings indicated that BD patients with anxiety symptoms have increased levels of inositol metabolism in the right PWM. Furthermore, the level of membrane phospholipid catabolism in the left PWM of BD patients with anxiety symptoms decreased with increasing age and onset age. Our results provide some references for the pathophysiological mechanism in BD patients with anxiety symptoms.

Molecular cloning, characterization and expression analysis of CbPLDδ gene from Chorispora bungeana in low temperature

Chorispora bungeana (C. bungeana) is a rare alpine subnival species that is highly tolerant to low temperature stress. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid catabolism during plant growth and the stress response. In this study, one member of CbPLD gene family, CbPLDδ, was cloned from C. bungeana and was introduced into tobacco. This gene encodes an 864-amino acid protein with two catalytic HxKxxxxD motifs which are essential for phospholipase D activity. After the CbPLDδ gene is fused with the vector containing the GFP tag, subcellular localization showed that CbPLDδ was predominately located in the cell membrane. RT-qPCR and histochemical GUS assays showed that CbPLDδ gene was induced by low temperature and expressed predominantly in leaf and root. Compared with wild-type tobacco, CbPLDδ transgenic tobacco showed higher activities of antioxidant enzymes, and lower levels of malonidiadehyde and electrolyte leakage under low temperature stress. These results reflected that CbPLDδ is involved in the response to low temperature stress, and has the potential to improve the low temperature tolerance of plants.

Transcriptomic response to changing ambient phosphorus in the marine dinoflagellate Prorocentrum donghaiense.

Dinoflagellates represent major contributors to the harmful algal blooms in the oceans. Phosphorus (P) is an essential macronutrient that limits the growth and proliferation of dinoflagellates. However, the specific molecular mechanisms involved in the P acclimation of dinoflagellates remain poorly understood. Here, the transcriptomes of a dinoflagellate Prorocentrum donghaiense grown under inorganic P-replete, P-deficient, and inorganic- and organic P-resupplied conditions were compared. Genes encoding low- and high-affinity P transporters were significantly down-regulated in the P-deficient cells, while organic P utilization genes were significantly up-regulated, indicating strong ability of P. donghaiense to utilize organic P. Up-regulation of membrane phospholipid catabolism and endocytosis provided intracellular and extracellular organic P for the P-deficient cells. Physiological responses of P. donghaiense to dissolved inorganic P (DIP) or dissolved organic P (DOP) resupply exhibited insignificant differences. However, the corresponding transcriptomic responses significantly differed. Although the expression of multiple genes was significantly altered after DIP resupplementation, few biological processes varied. In contrast, various metabolic processes associated with cell growth, such as translation, transport, nucleotide, carbohydrate and lipid metabolisms, were significantly altered in the DOP-resupplied cells. Our results indicated that P. donghaiense evolved diverse DOP utilization strategies to adapt to low P environments, and that DOPs might play critical roles in the P. donghaiense bloom formation.

Mitochondrial uncoupling links lipid catabolism to Akt inhibition and resistance to tumorigenesis.

To support growth, tumour cells reprogramme their metabolism to simultaneously upregulate macromolecular biosynthesis while maintaining energy production. Uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is uncoupled from ATP synthesis, resulting in nutrient wasting. Here using a UCP3 transgene targeted to the basal epidermis, we show that forced mitochondrial uncoupling inhibits skin carcinogenesis by blocking Akt activation. Similarly, Akt activation is markedly inhibited in UCP3 overexpressing primary human keratinocytes. Mechanistic studies reveal that uncoupling increases fatty acid oxidation and membrane phospholipid catabolism, and impairs recruitment of Akt to the plasma membrane. Overexpression of Akt overcomes metabolic regulation by UCP3, rescuing carcinogenesis. These findings demonstrate that mitochondrial uncoupling is an effective strategy to limit proliferation and tumorigenesis through inhibition of Akt, and illuminate a novel mechanism of crosstalk between mitochondrial metabolism and growth signalling.

Phospholipase Dα from Chorispora bungeana: cloning and partial functional characterization

Chorispora bungeana Fisch. and C.A. Mey (C. bungeana) is a rare alpine subnival plant species that is highly tolerant to environmental stress. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid catabolism during plant growth, development and stress responses. We have isolated and partially characterized a full-length cDNA encoding PLDα from the plantlets in vitro of C. bungeana, with the aim of furthering our understanding of the role of PLDα at the molecular level. The C. bungeana PLDα (CbPLDα) was found to encode a 810-amino acid protein with moderate to high nucleotide sequence similarity to previously reported plant PLDα genes. RT-PCR analyses suggested that CbPLDα gene expression is correlated with the CbPLDα activity when C. bungeana plantlets were treated with low temperature (4, 0 and −4 °C). Furthermore, the CbPLDα gene was found to be expressed at high levels in leaf and root tissues. These results indicate that the CbPLDα may play an important role in response to low temperature stresses in C. bungeana.

Characterization of Phospholipase D from Arabidopsis thaliana Callus in Response to Ent-Kaurene Diterpenoid Leukamenin E

A variety of components have been isolated from various higher plants and characterized as allelochemicals, which can play an important role in natural plant communities. Leukamenin E is an ent-kaurene diterpenoid isolated from Isodon racemosa (Hemsl) Hara. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid catabolism during plant growth, development, and stress responses. To further explore and elaborate the responses of PLD to leukamenin E treatment, the activities and expression patterns of the PLD gene in Arabidopsis thaliana (A. thaliana) callus were researched. When A. thaliana callus was incubated with leukamenin E at concentrations of 100 and 200 μM for 48 h, the activities of PLD in microsomal and mitochondrial membranes exhibited an upregulation behavior, with the highest levels at 24 and 36 h, respectively. RT-PCR analyses suggested that PLD activity partially corresponded to the A. thaliana PLD gene transcript level. As indicators of membrane damage, electrolyte leakage and malondialdehyde contents increased significantly and peaked at hour 36 and then decreased when A. thaliana callus was treated by leukamenin E. The contents of osmotic adjustment components proline and soluble sugar also shared similar trends. These results demonstrated that the specific mechanism of the A. thaliana response to leukamenin E was linked to PLD.

Humanized-Single Domain Antibodies (VH/VHH) that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA2). The PLA2 exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/VHH) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-VHH, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH purified from the E. coli homogenates neutralized PLA2 enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/VHH covered the areas around the PLA2 catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/VHH would ameliorate/abrogate the principal toxicity of the venom PLA2 (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

Indexation of cerebral cell membrane phospholipid catabolism by the non-invasively determined cerebral 31-phosphorus neurospectroscopic phosphodiester peak

Breakdown of mammalian cerebral cell membrane phospholipids releases phosphorylated polar head groups from the sn-3 phospholipid position, including phosphorylcholine and phosphorylethanolamine. Glycerophosphorylcholine and glycerophosphorylethanolamine are on their catabolic pathways and have been assigned to the phosphodiester narrow resonance obtained from 31-phosphorus neurospectroscopy, accounting for approximately 38% of the overall signal; therefore in human in vivo 31-phosphorus neurospectroscopy neuropsychiatric studies this narrow resonance has been used to index the catabolism of cerebral cell membrane phospholipids non-invasively. However, for ethical reasons direct assessment of this assumption has not hitherto been possible in humans. Recently, it has become possible to analyze signal directly from the cell membrane motion-restricted phospholipids by analysis of a broad resonance signal. It was therefore hypothesized that there should be a negative correlation between the phosphodiester narrow resonance and the broad resonance signal if the former does indeed index cell membrane phospholipid catabolism. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 54 human subjects (mean age 38 years; 41 male), including normal volunteers and patients with schizophrenia, in order potentially to widen the range of phosphodiester and broad resonance values. Spectra were obtained from 70 × 70 × 70 mm 3 voxels using an image-selected in vivo spectroscopy pulse sequence. There was a highly significant negative correlation between the phosphodiester resonances and the broad resonance signals ( r = −0.509, P < 0.0001). This result is consistent with the hypothesis that the phosphodiester narrow resonance does index cell membrane phospholipid catabolism in non-invasive human neuropsychiatric studies.

Molecular cloning and partial characterization of a novel phospholipase D gene from Chorispora bungeana

Chorispora bungeana Fisch. and C.A. Mey (Chorispora bungeana) is a rare alpine subnival plant species that is highly tolerant to environmental stress. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid catabolism during plant growth, development and stress responses. We have isolated and partially characterized a full-length cDNA encoding PLD from the calluses of Chorispora bungeana (C. bungeana), with the aim of furthering our understanding of the role of PLD at the molecular level. The C. bungeana PLD was found to encode a 903-amino acid protein with moderate to high nucleotide sequence similarity to previously reported plant PLD beta genes. The predicted CbPLD sequence showed the characteristic C2 domain and the phospholipase domains conferring calcium sensitivity and the enzyme activity, respectively. The transcripts of CbPLD accumulated highly when Chorispora bungeana was treated with cold (4 and −4°C), abscisic acid (ABA), salinity, hydrogen peroxide (H2O2) and heat. These results indicate that the CbPLD may play an important role in response to stresses in Chorispora bungeana.

Immunohistochemical localization of phospholipase D in strawberry (Fragaria ananassa Duch.) fruits

Phospholipase D, the key enzyme involved in membrane phospholipid catabolism, was localized in strawberry (Fragaria ananassa Duch.) fruits during development using immunohistochemical techniques. Typical structural features such as the nucleus, cell wall, plasma membrane, mitochondria and vacuole were noticeable in cells of young strawberry fruits. Cytoplasmic structural features were less pronounced in fully grown and turning red fruits, except cell wall, which was considerably enlarged. Also, extensive microvesiculation from the plasma membrane was noticeable in ripening fruits. The number of visible gold particles indicative of phospholipase D increased during development. Phospholipase D was found to be associated with membranous structures and vacuole. Interestingly, gold-particles could also be observed in the cell wall space in ripening fruits suggesting that cytosolic contents may have leaked into the cell wall space as a result of membrane deterioration and loss of compartmentalization. The role of phospholipase D during development and ripening of strawberry, a non-climacteric fruit, is discussed.

Proton and 31-phosphorus neurospectroscopy in the study of membrane phospholipids and fatty acid intervention in schizophrenia, depression, chronic fatigue syndrome (myalgic encephalomyelitis) and dyslexia

SummaryNeurospectroscopy allows biochemical processes in the brain to be studied non-invasively. At magnetic field strengths of 1.5 T or higher, cerebral proton neurospectroscopy allows the ascertainment of values of myo-inositol, choline-containing compounds, creatine, glutamate, glutamine, and N-acetyl aspartate. At similar field strengths, cerebral 31-phosphorus neurospectroscopy allows the ascertainment of values of phosphomonoesters, inorganic phosphate, phosphodiesters, phosphocreatine, and the gamma, alpha and beta nucleotide triphosphate (mainly adenosine triphosphate) resonances. Since choline is a common polar head group at the Sn3 position of membrane phospholipid molecules, a raised level of free choline, as indexed by proton neurospectroscopy, can indicate relatively low anabolism of membrane phospholipid molecules. Furthermore, the choline peak includes phosphorylcholine and glycerophosphorylcholine and even ethanolamine. The phosphomonoesters peak measured using 31-phosphorus spectroscopy includes major contributions from phosphocholine, phosphoethanolamine and L-phosphoserine, which are important precursors of membrane phospholipids, while the phosphodiesters peak includes contributions from glycerophosphocholine and glycerophosphoethanolamine, which are important products of membrane phospholipid catabolism. Hence proton neurospectroscopy and 31-phosphorus neurospectroscopy can yield important information relating to the metabolism of cerebral membrane phospholipids. The application of these techniques to the investigation of membrane phospholipid metabolism in schizophrenia, depression, chronic fatigue syndrome (myalgic encephalomyelitis or M.E.) and dyslexia is described.

Alterations of phospholipid metabolites in postmortem brain from patients with Alzheimer's disease.

To test the hypothesis that brain cell membranes degenerate in Alzheimer's disease (AD), we measured the levels of phospholipids, their water-soluble metabolites, and glycerophosphocholine (GPC) cholinephosphodiesterase activity in postmortem brain tissue from patients with AD and age-matched controls. We found significantly higher levels of the phospholipid catabolite GPC in AD brain. In contrast, choline and ethanolamine levels were significantly lower in AD, and phospholipid levels were slightly decreased. Furthermore, in AD the activity of the GPC-degrading enzyme GPC cholinephosphodiesterase was unaltered. Our results indicate that membrane phospholipid catabolism is increased in AD brain. Inasmuch as the tissue levels of initial phospholipid precursors were decreased, we suggest that phospholipid turnover is elevated in this neurodegenerative disease.

Regulation of membrane phospholipid catabolism in senescing carnation flowers

Evidence has been obtained for the involvement of μM levels of Ca2+ in phospholipid catabolism during petal senescence by following the breakdown of [U‐14C]‐phosphatidylcholine by microsomal membranes from cut carnation (Dianthus caryophyllus L. cv. White‐sim) flowers. Phospholipid degradation was mediated by three membrane‐associated lipases, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4) and lipolytic acyl hydrolase. The activities of phospholipase D and phosphatidic acid phosphatase were stimulated by 30 and 100%, respectively, in the presence of 40 μM free Ca2+, and the Ca2+‐stimulation of phosphatidic acid phosphatase was calmodulin‐dependent. When L‐3‐phosphatidyl‐[2‐3H]‐inositol and L‐3‐phosphatidyl‐[N‐methyl‐3H]‐choline were used as substrates, inositol and choline accounted for 95 and 99%, respectively, of the water‐soluble radiolabelled products. This suggests a predominance of phospholipase D activity over phospholipase C activity in these membranes.Breakdown of membrane phospholipids in senescing carnations is known to be accelerated by treatment of young flowers with ethylene. To determine whether this involves a specific turnover of phosphatidylinositol as observed in animal systems in response to certain agonists, young flowers pre‐labelled with 32PO3‐4 were treated with 10 ppm ethylene. All phospholipids incorporated the label, but no enhanced turnover of phosphatidylinositol was observed. Inositol 1,4,5‐triphosphate did not release Ca2+ from preloaded microsomal vesicles at concentrations known to be effective in animal systems (i.e. &lt; 5 μM) although release of Ca2+ was observed when a higher (20 μM) concentration was used.

Membrane phospholipid catabolism and Ca2+ activity in control of senescence

Leshem, Y. Y. 1987. Membrane phospholipid catabolism and Ca2+ activity in control of senescence.A key role in the regulation of plant development and senescence appears to be a finely balanced equilibrium between membrane phospholipid catabolism on the one hand, and synthesis and remodelling on the other. In the catabolic “phosphatidyl‐linoleyl(‐enyl) cascade”, entering of Ca2+ into the cytosol triggers the catabolic process by binding to calmodulin and activating phospholipase A2, (EC 3.1.1.4). The latter proceeds to release linoleic or linolenic acid from the sn‐2 (stereospecific numbering) location of intact phospholipid, thus providing substrate for lipoxygenase (EC 1.13.11.12). The action of lipoxygenase then generates a series of oxy‐free radicals, ethylene, endogenous Ca2+ ionophores, malondialdehyde and jasmonic acid. These may recycle to the membrane, causing the entry of more Ca2+ and induction of a further, identical catabolic cycle. With increased cycling, membranes become progressively senescent and undergo biophysical changes altering microviscosity, fluidity, phase configurations of membrane phospholipids and transition temperatures. The cascade does not appear to be specific for the phospholipid substrate, and it is envisaged that besides phospholipase A2, both phospholipase B (EC 3.1.1.5) and lipolytic acylhydrolase could participate in the process.A parallel process counteracting the above, is membrane remodelling and turnover, proceeding initially by the same Ca2+‐ and possibly calmodulin‐triggering, but leading via phospholipase C (EC 3.1.4.10) action and diacylglycerol formation to protein kinase activation and proton pump recharging. It is speculated that auxin and cytoki‐nin, albeit by different pathways, induce this route, for which membrane phospho‐inositides may be the preferred membrane‐associated phospholipid substrate.

Accumulation of lysophosphoglycerides with arrhythmogenic properties in ischemic myocardium.

Lysophosphoglycerides, products of membrane phospholipid catabolism known to influence membrane function in several systems, appeared in the effluents of anoxic isolated rabbit hearts perfused at low flow and accumulated in perfused hearts and myocardium rendered ischemic in situ. Comparable concentrations of lysophosphoglycerides bound to albumin markedly and reversibly altered action potentials of isolated canine Purkinje fibers in vitro. Changes induced included diminution of the maximum diastolic potential, peak dV/dt of phase zero, amplitude, and action potential duration--alterations resembling those seen in ischemic myocardium in vivo. These electrophysiological alterations are compatible with changes implicated in predisposing to dysrhythmia dependent on reentry, a phenomenon potentiated by the presence of zones of decreased conduction. Thus, accumulation of lysophosphoglycerides induced by ischemia may contribute to the genesis of malignant dysrhythmia early after its onset.