Hypoxia-induced GPCPD1 depalmitoylation triggers mitophagy via regulating PRKN-mediated ubiquitination of VDAC1.

Mitophagy, which selectively eliminates the dysfunctional and excess mitochondria by autophagy, is crucial for cellular homeostasis under stresses such as hypoxia. Dysregulation of mitophagy has been increasingly linked to many disorders including neurodegenerative disease and cancer. Triple-negative breast cancer (TNBC), a highly aggressive breast cancer subtype, is reported to be characterized by hypoxia. However, the role of mitophagy in hypoxic TNBC as well as the underlying molecular mechanism is largely unexplored. Here, we identified GPCPD1 (glycerophosphocholine phosphodiesterase 1), a key enzyme in choline metabolism, as an essential mediator in hypoxia-induced mitophagy. Under the hypoxic condition, we found that GPCPD1 was depalmitoylated by LYPLA1, which facilitated the relocating of GPCPD1 to the outer mitochondrial membrane (OMM). Mitochondria-localized GPCPD1 could bind to VDAC1, the substrate for PRKN/PARKIN-dependent ubiquitination, thus interfering with the oligomerization of VDAC1. The increased monomer of VDAC1 provided more anchor sites to recruit PRKN-mediated polyubiquitination, which consequently triggered mitophagy. In addition, we found that GPCPD1-mediated mitophagy exerted a promotive effect on tumor growth and metastasis in TNBC both in vitro and in vivo. We further determined that GPCPD1 could serve as an independent prognostic indicator in TNBC. In conclusion, our study provides important insights into a mechanistic understanding of hypoxia-induced mitophagy and elucidates that GPCPD1 could act as a potential target for the future development of novel therapy for TNBC patients.Abbreviations: ACTB: actin beta; 5-aza: 5-azacytidine; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; ChIP: chromatin immunoprecipitation; co-IP: co-immunoprecipitation; CQ: chloroquine; CsA: cyclosporine; DOX: doxorubicin; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GPCPD1: glycerophosphocholine phosphodiesterase 1; HAM: hydroxylamine; HIF1A: hypoxia inducible factor 1 subunit alpha; HRE: hypoxia response element; IF: immunofluorescence; LB: lysis buffer; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; LC-MS: liquid chromatography-mass spectrometry; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; MDA231: MDA-MB-231; MDA468: MDA-MB-468; MFN1: mitofusin 1; MFN2: mitofusin 2; MKI67: marker of proliferation Ki-67; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; OS: overall survival; PalmB: palmostatin B; PBS: phosphate-buffered saline; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; SDS: sodium dodecyl sulfate; TOMM20: translocase of outer mitochondrial membrane 20; TNBC: triple-negative breast cancer; VBIT-4: VDAC inhibitor; VDAC1: voltage dependent anion channel 1; WT: wild type.

The alteration of the expression level of neuropathy target esterase in human neuroblastoma SK-N-SH cells disrupts cellular phospholipids homeostasis.

Neuropathy target esterase (NTE) has been proven to act as a lysophospholipase (LysoPLA) and phospholipase B (PLB) in mammalian cells. In this study, we took human neuroblastoma SK-N-SH cells as the research object and explored the effect of NTE on phospholipid homeostasis. The results showed that phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) levels significantly increased (> 40%), while glycerophosphocholine (GPC) decreased (below 60%) after NTE gene was knockdown in the cells (NTE < 30% of control), which were prepared by gene silencing with dsRNA-NTE. However, in the NTE-overexpressed cells (NTE > 50% of control), which were prepared by expressing recombinant catalytic domain of NTE, LPC remarkably decreased (below 80%) and GPC enhanced (> 40%). Mipafox, a neuropathic organophosphorus compound (OP), significantly inhibited NTE-LysoPLA and NTE-PLB activities (> 95-99% inhibition at 50 µM), which was accompanied with a decreased GPC level (below 40%) although no change of the PC and LPC levels was observed; while paraoxon, a non-neuropathic OP, suppresses neither the activities of NTE-phospholipases nor the levels of PC, LPC, and GPC. Thus, we concluded that both the stable up- or down-regulated expression of NTE gene and the loss of NTE-LysoPLA/PLB activities disrupts phospholipid homeostasis in the cells although the inhibition of NTE activity only decreased GPC content without altering PC and LPC levels.

Protein crystallization promotes type 2 immunity and is reversible by antibody treatment.

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100.

In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique.

Clonorchis sinensis lysophospholipase inhibits TGF-ß1-induced expression of pro-fibrogenic genes through attenuating the activations of Smad3, JNK2, and ERK1/2 in hepatic stellate cell line LX-2.

Liver fibrosis is a wound healing response associated with chronic liver injury. Hepatic stellate cells (HSCs) activation is a key event in the development of liver fibrosis. Since helminths have the ability to live for decades in the host by establishing an adaptive relationship in the interplay with its hosts, we hypothesize that whether Clonochis sinensis LysophospholipaseA (CsLysoPLA), a component of excretory/secretory proteins, can attenuate the fibrogenic response by inhibiting activation of LX-2 cells, thereby balancing the pro-fibrotic and anti-fibrotic response during the Clonochis sinensis (C. sinensis) infection. In the present study, LX-2 cells were stimulated with CsLysoPLA in the presence of TGF-ß1, and the expressions of collagen type I (COL1A1), α-smooth muscle actin (α-SMA), and matrix metalloproteinase 2 (MMP2) were decreased. In addition, CsLysoPLA significantly inhibited the proliferation and migration of LX-2 cells stimulated by TGF-ß1. Pretreatment of LX-2 cells with CsLysoPLA attenuated the phosphorylation of Smad3 as well as JNK2 and ERK1/2 in response to the stimulation of TGF-ß1. For the first time, our results showed an anti-fibrogenic effect of CsLysoPLA by attenuating the response of LX-2 cells to TGF-ß1 through inhibiting the activations of Smad3, ERK1/2, and JNK2.

[The effects of Lysophospholipase from Clonorchis sinensis on the hepatic stellate cells and oval cells of rat].

AIM: To clarify the effects of the recombinant protein of Lysophospholipase from Clonorchis sinensis (CsLysoPLA) on the hepatic stellate cells (HSC) and oval cells of rat. METHODS: Binding of the recombinant CslysoPLA protein to the membrane of HSC and oval cells was identified by immunofluorescent staining. The HSC and oval cells were cultured and treated with the recombinant protein at different doses, and proliferation was quantified by MTT method. Cell cycle analysis was performed by flow cytometry. RESULTS: The recombinant CslysoPLA protein could bind to the membrane of HSC and oval cells. Compared to control, 2 mg/L and 20 mg/L the recombinant protein could promote HSC and oval cells growth (P<0.05), whereas 200 mg/L the recombinant protein could induce the cells necrosis, which associated with overt plasma membrane disruption. Oval cell number in G(2) phase of the recombinant protein 20 mg/L treated group was higher than that of control group. CONCLUSION: In vitro, the recombinant protein could induce HSC and oval cells proliferation at low concentrations (2 mg/L and 20 mg/L), whereas it also could induce the cells necrosis at high concentration (200 mg/L). These results suggested that CslysoPLA might play a role in the pathogenicity of C. sinensis.

Sphingosine 1-phosphate stimulates fibronectin matrix assembly through a Rho-dependent signal pathway.

Fibronectin matrix assembly is a cell-dependent process mediated by cell surface binding sites for the 70-kD N-terminal portion of fibronectin. We have shown that Rho-dependent cytoskeleton reorganization induced by lysophosphatidic acid (LPA) or the microtubule-disrupting agent nocodazole increases fibronectin binding (Zhang et al, Mol Biol Cell 8:1415, 1997). Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in mitogenesis and cytoskeletal remodelling. Both LPA and S1P are present in increased amounts in serum as compared with plasma as a result of platelet activation. Addition of S1P to human osteosarcoma MG63 cells or human foreskin fibroblasts increased cell-mediated binding and assembly of fibronectin. MG63 cells expressed the Edg-2 and Edg-4 G-protein-coupled receptors for bioactive lipids, whereas foreskin fibroblasts expressed Edg-2, Edg-3, and Edg-4. The stimulatory effect of S1P on the binding of fibronectin or the N-terminal 70-kD fragment of fibronectin was dynamic and due to increases in both the number and affinity of binding sites. The stimulation of 70-kD fragment binding by nanomolar S1P, like stimulation of binding by LPA or nocodazole, was blocked by inactivation of Rho with C3 exotoxin but not by pertussis toxin-mediated inactivation of Gi. These results indicate a common signal pathway leading to control of cellular fibronectin matrix assembly by bioactive lipids generated during blood coagulation.

Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production.

The implication in cholera toxin (CT) production of the newly identified gene, lypA, that encodes the lysophospholipase L2 of Vibrio cholerae, was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L2 activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of E1 Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae.

Lysophosphatidic acid-depleted serum, hepatocyte growth factor and stem cell growth factor stimulate colony growth of small cell lung cancer cells through a calcium-independent pathway.

Serum stimulates both Ca2+ mobilization and colony growth of many small cell lung cancer (SCLC) cell lines, but the factors involved remain unknown. We demonstrate that 1-oleoyl-lysophosphatidic acid (LPA), like serum, induced a dose-dependent increase in intracellular Ca2+ in the H-510, H-345, and H-69 SCLC cell lines with half maximal concentrations of 18 nM, 22 nM, and 20 nM, respectively. Two lines of evidence revealed that LPA was the major factor in serum responsible for mobilizing Ca2+ in these SCLC cell lines: (a) both LPA and serum exhibited cross desensitization in the Ca2+ mobilization assay; and (b) phospholipase B pretreatment of either LPA or serum prevented the ability of these agents to stimulate Ca2+ mobilization. In marked contrast, LPA at concentrations between 2 nM and 20 microM, unlike serum, failed to stimulate colony formation. Furthermore, phospholipase B treatment of serum did not inhibit serum-induced colony formation. We therefore searched for growth factors which could induce colony growth through a Ca(2+)-independent pathway. We found that both human recombinant hepatocyte growth factor and stem cell growth factor increased colony growth, but failed to stimulate an increase in intracellular Ca2+ in the H-510, H-345, and H-69 SCLC cell lines. Our results indicate that LPA-depleted serum, hepatocyte growth factor, and stem cell growth factor stimulate colony formation in SCLC cells through a Ca(2+)-independent pathway.

Lysophosphatidic acid mimics serum-induced sensitization of cyclic AMP accumulation.

Pretreatment of 1321N1 human astrocytoma cells with serum induces a pronounced increase in subsequent stimulation by forskolin and other agents of intracellular cyclic AMP accumulation, a phenomenon referred to as sensitization (Mol. Pharmacol. 39, 399-406, 1991). Pretreatment of these cells with lysophosphatidic acid induced sensitization to a similar extent as that with serum (approximately fivefold for forskolin stimulation and twofold for isoproterenol and prostaglandin E1 stimulation), with half-maximal effects at approximately 30 nM lysophosphatidic acid. Phosphatidic acid was effective but less potent whereas other lipids were ineffective. Sensitization by serum and by lysophosphatidic acid were almost completely inhibited by pertussis toxin pretreatment and partially inhibited by prolonged phorbol ester exposure to induce protein kinase C down-regulation. Among nine cell lines tested, those that exhibited sensitization with serum showed comparable sensitization with lysophosphatidic acid. The effects of both lysophosphatidic acid and serum were markedly inhibited by treatment with phospholipase B but only minimally altered with phospholipases A2, D, and C. Exposure of cells to phospholipase C alone induced approximately threefold sensitization, but both serum and lysophosphatidic acid were able to induce further three- to fourfold sensitization above that induced by phospholipase C alone. In contrast, the effects of serum and lysophosphatidic acid were not additive with each other. Together these results suggest that lysophosphatidic acid or a closely related compound present in serum is the factor responsible for sensitization of the cyclic AMP pathway.

Lysophosphatidic acid reverts the beta-adrenergic agonist-induced morphological response in C6 rat glioma cells.

Elevation of cAMP changes the morphology of C6 rat glioma cells from a fibroblast to an astrocyte type of appearance. This change is prevented by the presence of serum from different species (chicken, mouse, rat, horse, adult bovine, fetal bovine, and human) in the cell culture medium. In this communication the component in serum responsible for this effect is identified as lysophosphatidic acid for the following reasons: First, lysophosphatidic acid alone at concentrations which are present in serum reverts the morphological response. Second, both lysophosphatidic acid and the component in serum no longer revert the morphological response after treatment with phospholipase B (E.C.3.1.1.5). Third, lysophosphatidic acid and serum produce an analogous intracellular Cai2+ signal in rat glioma C6 cells as measured by fluorescence spectrophotometry with the Ca2+ ion indicator Fura 2. Fourth, both the morphological response and the Cai2+ increase are prevented by pretreatment of the cells with 100 ng/ml phorbol 12-myristate 13-acetate. Finally, a maximal Cai2+ increase induced by FCS prevents the subsequent Cai2+ signal by lysophosphatidic acid. Interestingly, the morphological response is also reverted by Al3+ together with F- ions and also by lower n-alkanols such as ethanol and n-propanol suggesting that a regulatory GTP-binding protein is involved in the reversion. It is further shown that activation of the phosphatidylinositol 4,5-bisphosphate cleavage signal system is not responsible for the reversion of the morphological response.

Lysophosphatidylcholine accumulation in ischemic human myocardium.

Lysophosphatidylcholine accumulates in the coronary sinus during pacing-induced myocardial ischemia in humans. This amphiphile accelerates Ca++ flux leading to cell injury in cultured cardiac myocytes, but it is not known whether lysophosphatidylcholine accumulation is injurious to human myocardium. In this study, we measured lysophosphatidylcholine in normal human myocardium obtained during cardiac surgery and exposed to ischemic conditions in vitro. Total lysophosphatidylcholine concentration (sum of lysophosphatidylcholine remaining in tissue and lysophosphatidylcholine released into the buffer) increased from 0.73 +/- 0.08 nmol/mg protein at baseline to 1.83 +/- 0.45 nmol/mg protein after 5 minutes of ischemia (p < 0.001), and was associated with evidence of cell injury (26% depletion of tissue lactate dehydrogenase). Significant lysophosphatidylcholine release into the incubation buffer (0.41 +/- 0.11 nmol/mg protein) also occurred after 5 minutes of ischemia. In contrast, there was no lysophosphatidylcholine accumulation or release and no lactate dehydrogenase depletion in oxygenated and perfused controls. Attenuation of lysophosphatidylcholine accumulation by incubation with lysophospholipase did not prevent cell injury. Lysoplasmalogen was not detected in ischemic tissue. We conclude that lysophosphatidylcholine accumulation is a marker of myocardial ischemia in humans.

Antimicrobial activities of clofazimine and B669 are mediated by lysophospholipids.

The susceptibilities of a range of gram-positive and gram-negative microbial pathogens to clofazimine and its analog B669 (0.1 to 32 micrograms/ml), as well as the effects of these agents on membrane phospholipid metabolism in Staphylococcus aureus and Escherichia coli, have been investigated in vitro. Gram-positive bacteria were found to be generally susceptible to these agents, whereas gram-negative organisms were uniformly resistant. Exposure of S. aureus to both agents (1 to 5 micrograms/ml), especially B669, caused dose-related enhancement of the activity of phospholipase A2, according to an increase in the release of 3H-radiolabeled arachidonate and lysophosphatidylethanolamine ([3H]LPE) from bacterial-membrane phospholipids. Treatment of E. coli with the riminophenazines also increased the release of [3H]arachidonate and [3H]LPE. Growth of gram-positive but not gram-negative bacteria was inhibited by LPE and lysophosphatidylcholine. Moreover, coincubation with alpha-tocopherol (vitamin E), a lysophospholipid complex-forming agent, or with lysophospholipase protected gram-positive bacteria against the riminophenazines as well as against lysophospholipids. The results from this study are consistent with a mechanism whereby lysophospholipids mediate the activities of the two drugs.

Regulation of [3H]nitrendipine binding by phospholipases A2 and C through direct and GTP-sensitive mechanisms.

We compared the effects of Phospholipases A2, C, B and D on [3H]nitrendipine binding to hamster cardiac membranes, in the absence and presence of ATP or GTP. Phospholipase A2, competitively inhibited [3H]nitrendipine binding to hamster cardiac membranes unchanged by ATP or GTP (Ki = 5 ng/ml); as evidenced by complete and reversible displacement of [3H]nitrendipine binding and increase in KD on Scatchard analyses. Phospholipase C also completely inhibited [3H]nitrendipine binding to hamster cardiac membranes (Ki = 5 micrograms/ml) with a decrease in Bmax and no change in KD on Scatchard analyses. The addition of GTP alone inhibited the PLC effect in EGTA-treated membranes. The addition of GTP with either CaCl2 or ATP or both resulted in an equal and opposite enhancement of the PLC effect. Phospholipases B and D had no effect on [3H]nitrendipine binding. These data support: (1) Direct effect of PLA2 on dihydropyridine binding. (2) Indirect regulation of dihydropyridine binding by Phospholipase C through a GTP and ATP-sensitive mechanism.

[Purification and properties of lysophospholipase from the venom of the giant hornet Vespa orientalis]. / Ochistka i svoistva lizofosfolipazy iz aida bol'shogo shershnia Vespa orientalis.

Using biospecific chromatography on polylysocephamide, a toxic phospholipase possessing a presynaptic effect on neuromuscular preparations was isolated from the venom of the giant hornet Vespa orientalis. The enzyme was shown to possess a high hydrolytic activity towards 1-acyllysophosphatidylcholine within a narrow pH range (pH optimum 7.5). The enzyme activity was suppressed by detergents of various chemical composition. Lysophospholipase caused an intensive hemolysis of washed human erythrocytes. The catalytic and hemolytic functions of the enzyme were sensitive to metal ions, however, in a different degree. Ca2+ and Mn2+ activated, while Cu2+ and Zn2+ inhibited the enzyme. Mg2+ and Sr2+ had no effect on the enzyme activity.