Accumulation Of Phosphatidylinositol Research Articles

De Novo Synthesis of Perdeuterated Phosphoinositide by Installing a Non-native Phospholipid Biopathway in E. coli.

Phosphatidylinositol (PI) and its phosphorylated derivatives are of paramount importance in cellular functions and diseases. Understanding their diverse roles is, however, challenged by difficulties in synthesis and labeling techniques. In this proof-of-concept study, we demonstrate that PI can be straightforwardly de novo-synthesized and deuterium (2H)-labeled in Escherichia coli by genomic insertion of PI synthase from Trypanosoma brucei under constitutive synthetic promoter proD. Insertion into loci atpi-gidB and ybb revealed PI accumulation of 41% and 34% (mol/mol), respectively, when cultivated with glycerol as the sole carbon source. Growth of the atpi-gidB-PIS strain in deuterium-labeled (2H) substrates D2O, D8-glycerol, and D6-myo-inositol achieved PI deuteration of 90%, PE deuteration of 95%, and total fatty acids|fatty acid (FA) deuteration of 97%. This study offers an alternative convenient route to chemical and enzymatic labeling synthesis of PI; more excitingly, this work also, in principle, opens a door for tailoring the FA profile of deuterated PI/PE for task-specific application by repurposing FA biosynthesis pathways.

Biogenesis of Outer Membrane Vesicles Concentrates the Unsaturated Fatty Acid of Phosphatidylinositol in Capnocytophaga ochracea.

Bacterial outer membrane vesicles (OMVs) are spherical lipid bilayer nanostructures released by bacteria that facilitate oral biofilm formation via cellular aggregation and intercellular communication. Recent studies have revealed that Capnocytophaga ochracea is one of the dominant members of oral biofilms; however, their potential for OMV production has yet to be investigated. This study demonstrated the biogenesis of OMVs in C. ochracea associated with the concentration of unsaturated fatty acids of phosphatidylinositol (PI) and characterized the size and protein profile of OMVs produced at growth phases. Transmission electron microscopy showed isolated spherical structures from cells stained with heavy metals, indicating the production of OMVs with a size ranging from 25 to 100 nm. Lipidome analysis revealed the presence of phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, and PI as the main lipids. Some unsaturated fatty acids of PI were present specifically in OMV and little in the outer membrane, suggesting that OMVs are generated from a specific region of the membrane through blebbing rather than a random process such as cell lysis. Furthermore, the lack of similar PI accumulation in the OMV of Porphyromonas gingivalis suggests that C. ochracea has a different biogenesis mechanism. The blebbing mechanism was further supported by higher OMV production occurring at the exponential phase in comparison to the stationary phase, where cell lysis is more likely to occur. Further, comparative protein profile of OMVs isolated under different growth phases may indicate that the OMV cargo does not largely vary with growth phases. The present study provides a basis for further understanding the roles of C. ochracea OMVs in oral biofilms as well as systemic diseases that C. ochracea involves.

Imaging cholesterol depletion at the plasma membrane by methyl-β-cyclodextrin

The most common method to modify cellular cholesterol (Chol) content in vitro is to incubate cells with cyclodextrin (CD). However, it is not well understood how Chol is removed by CD. Recent development of protein probes that bind Chol allowed to visualize and to follow cellular Chol semi-quantitatively. In this image, non-toxic Chol-binding D4 fragment of bacterial toxin, perfringolysin O was conjugated with mCherry (mCherry-D4) (1Abe M. Makino A. Hullin-Matsuda F. Kamijo K. Ohno-Iwashita Y. Hanada K. Mizuno H. Miyawaki A. Kobayashi T. A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol 4,5-bisphosphate to the cleavage furrow during cytokinesis.Mol. Cell Biol. 2012; 32: 1396-1407Crossref PubMed Scopus (98) Google Scholar) and the protein was transiently expressed in HeLa cells for 48 h. Cells were synchronized to mitosis and incubated with and without 1 mM methyl-b-CD (MbCD) in the presence of EGFP-D4 in the medium. Selected images are shown. Accumulated images are shown in Supplemental videos 1 and 2. Average fluorescence intensities of EGFP-D4 (green) and mCherry-D4 (magenta) were quantified in the area surrounded by the white rectangle and schematically shown in the kymographs. The quantitated data are shown as graphs in Supplemental Fig. 1. Supplemental Fig. 2 shows that equivalent amount of EGFP-D4 and mCherry-D4 displayed similar Chol concentration dependent binding to phosphatidylcholine/Chol membranes. Under this condition, mCherry-D4 and EGFP-D4 bind accessible Chol (2Das A. Brown M.S. Anderson D.D. Goldstein J.L. Radhakrishnan A. Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis.Elife. 2014; 3e02882Crossref Scopus (171) Google Scholar) in the inner and outer leaflet of the plasma membrane, respectively (1Abe M. Makino A. Hullin-Matsuda F. Kamijo K. Ohno-Iwashita Y. Hanada K. Mizuno H. Miyawaki A. Kobayashi T. A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol 4,5-bisphosphate to the cleavage furrow during cytokinesis.Mol. Cell Biol. 2012; 32: 1396-1407Crossref PubMed Scopus (98) Google Scholar). Due to high threshold of Chol-detection by D4, intracellular organelles were not labeled with mCherry-D4 and depletion of Chol was accompanied by the intracellular accumulation of the protein. MbCD induced the rapid decrease (5-15 min) of inner leaflet Chol below the D4-binding threshold followed by the slow depletion phase in both outer and inner leaflets. The interaction between D4 or MbCD and cultured cells is not fully elucidated. Although MbCD is endocytosed, endocytosis is inhibited in mitotic cells (3Kobayashi T. Pagano R.E. Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface.J. Biol. Chem. 1989; 264: 5966-5973Abstract Full Text PDF PubMed Google Scholar). Binding of acrylodan-labeled D4 to PC/Chol membrane is inhibited by cytosol (4Courtney K.C. Fung K.Y. Maxfield F.R. Fairn G.D. Zha X. Comment on 'Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol'.Elife. 2018; 7e38493Crossref PubMed Scopus (16) Google Scholar). In addition to Chol, MbCD is reported to interact with other lipids and protein components of the membrane. However, extraction of cytosolic protein by MbCD has not been reported. Our result suggests that the removal of outer leaflet Chol by MbCD is accompanied by the rapid flip of Chol from the inner leaflet to the outer leaflet. However, we cannot exclude the possibility that MbCD preferentially removed Chol from the inner leaflet. EQUIPMENT: FV 1000 confocal microscope with a 60x 1.1 NA Plan Apo objective lens (Olympus) equipped with an environmental chamber maintained with humidity, 37°C, and 5% CO2. ImageQuant LAS 500 (Cytiva). REAGENTS: DMEM supplemented with 10% fetal bovine serum, Lipofectamine 3000 (Thermo Fisher Scientific), pET28/His6-EGFP-D4 (RIKEN BRC, Gene Engineering Division, Catalog # RDB13961), pET28/His6-mCherry-D4 (RIKEN BRC, Catalog # RDB14300), pDEST/mCherry-D4 (1), nocodazole (Sigma-Aldrich), MβCD (Cyclolab), SYPRO Ruby (Thermo Fisher Scientific). This article contains supplemental data. The authors declare that they have no conflicts of interest with the contents of this article. We are grateful to Dr. Françoise Hullin-Matsuda from INSA Lyon for critically reading the manuscript. M. A. conceptualization, methodology, investigation, data curation, writing-original draft preparation, reviewing, and editing. T. K. conceptualization, writing-original draft preparation, reviewing, and editing.

Inositol monophosphatase 1 as a novel interacting partner of RAGE in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation end products (RAGE) plays an important role in PAH by promoting proliferation of pulmonary vascular cells. RAGE is also known to mediate activation of Akt signaling, although the particular molecular mechanism remains unknown. This study aimed to identify the interacting partner of RAGE that could facilitate RAGE-mediated Akt activation and vascular remodeling in PAH. The progressive angioproliferative PAH was induced in 24 female Sprague-Dawley rats ( n = 8/group) that were randomly assigned to develop PAH for 1, 2, or 5 wk [right ventricle systolic pressure (RVSP) 56.5 ± 3.2, 63.6 ± 1.6, and 111.1 ± 4.5 mmHg, respectively, vs. 22.9 ± 1.1 mmHg in controls]. PAH triggered early and late episodes of apoptosis in rat lungs accompanied by RAGE activation. Mass spectrometry analysis has identified IMPA1 as a novel PAH-specific interacting partner of RAGE. The proximity ligation assay (PLA) confirmed the formation of RAGE/IMPA1 complex in the pulmonary artery wall. Activation of IMPA1 in response to increased glucose 6-phosphate (G6P) is known to play a critical role in inositol synthesis and recycling. Indeed, we confirmed a threefold increase in G6P ( P = 0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), membrane translocation of PI3K, and a threefold increase in membrane Akt levels ( P = 0.02) and Akt phosphorylation. We conclude that the formation of the newly discovered RAGE-IMPA1 complex could be responsible for the stimulation of inositol pathways and activation of Akt signaling in PAH.

Effect of tamoxifen on the sphingolipid biosynthetic pathway in the different intraerythrocytic stages of the apicomplexa Plasmodium falciparum

Parasites of the genus Plasmodium responsible for Malaria are obligate intracellular pathogens residing in mammalian red blood cells, hepatocytes, or mosquito midgut epithelial cells. Regarding that detailed knowledge on the sphingolipid biosynthetic pathway of the apicomplexan protozoan parasites is scarce, different stages of Plasmodium falciparum were treated with tamoxifen in order to evaluate the effects of this drug on the glycosphingolipid biosynthesis. Thin layer chromatography, High performance reverse phase chromatography and UV-MALDI-TOF mass spectrometry were the tools used for the analysis. In the ring forms, the increase of NBD-phosphatidyl inositol biosynthesis was notorious but differences at NBD-GlcCer levels were undetectable. In trophozoite forms, an abrupt decrease of NBD-acylated GlcDHCer and NBD-GlcDHCer in addition to an increase of NBD-PC biosynthesis was observed. On the contrary, in schizonts, tamoxifen seems not to be producing substantial changes in lipid biosynthesis. Our findings indicate that in this parasite, tamoxifen is exerting an inhibitory action on Glucosylceramidesynthase and sphingomyelin synthase levels. Moreover, regarding that Plasmodium does not biosynthesize inositolphosphoceramides, the accumulation of phosphatidylinositol should indicate an inhibitory action on glycosylinositol phospholipid synthesis.

5'-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion.

Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5'-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2-Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease.

The PTEN tumor suppressor gene and its role in lymphoma pathogenesis.

The phosphatase and tensin homolog gene PTEN is one of the most frequently mutated tumor suppressor genes in human cancer. Loss of PTEN function occurs in a variety of human cancers via its mutation, deletion, transcriptional silencing, or protein instability. PTEN deficiency in cancer has been associated with advanced disease, chemotherapy resistance, and poor survival. Impaired PTEN function, which antagonizes phosphoinositide 3-kinase (PI3K) signaling, causes the accumulation of phosphatidylinositol (3,4,5)-triphosphate and thereby the suppression of downstream components of the PI3K pathway, including the protein kinase B and mammalian target of rapamycin kinases. In addition to having lipid phosphorylation activity, PTEN has critical roles in the regulation of genomic instability, DNA repair, stem cell self-renewal, cellular senescence, and cell migration. Although PTEN deficiency in solid tumors has been studied extensively, rare studies have investigated PTEN alteration in lymphoid malignancies. However, genomic or epigenomic aberrations of PTEN and dysregulated signaling are likely critical in lymphoma pathogenesis and progression. This review provides updated summary on the role of PTEN deficiency in human cancers, specifically in lymphoid malignancies; the molecular mechanisms of PTEN regulation; and the distinct functions of nuclear PTEN. Therapeutic strategies for rescuing PTEN deficiency in human cancers are proposed.

Abstract 4959: High-resolution MALDI imaging mass spectrometry demonstrated the correlation between phosphatidylinositol profiles and invasion/metastasis in human breast cancer

Abstract High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI IMS) is an emerging application for lipid research that provides a detailed histological distribution of ionized lipids within tissue section. Phosphatidylinositol (PI) is one of the major classes of membrane phospholipid. The cellular membrane fluidity and signal transduction activity could be modulated by the alteration of fatty acid composition of PIs. Especially, PI-3kinase pathway, which can be activated by the phosphorylation of PIs, has become a great concern in breast cancer treatment. Although the amount of PIs is known to increase in human breast cancer tissues, it remains unclear how PIs are distributed in cancerous tissues. In this study, we investigated the detailed spatial distribution of PIs in breast tissues obtained from 24 patients, including 23 invasive ductal carcinoma (IDC), 4 ductal carcinoma in situ (DCIS), and 4 fibrocystic change lesions by using HR-MALDI IMS. The distribution of PIs was visualized at the resolution of 10um. 10 PIs with different fatty acid composition were detected in breast tissues. High-resolution analysis revealed the fatty acid composition of PIs was altered specifically in cancer cell clusters compared to stromal cells and non-malignant epithelia. PIs carrying monounsaturated fatty acid (MUFA) and PIs carrying polyunsaturated fatty acid (PUFA) showed the specific localization to cancer cell clusters. However, the proportion of PIs carrying MUFA and PIs carrying PUFA showed a clear inverse correlation in cancer cells. When we compared the PI profiles of IDC, DCIS, and FC, it was suggested that the accumulation of PIs carrying PUFA was occurred during the step of invasion. Moreover, lymph node metastasis well correlated with the accumulation of PIs carrying PUFA in cancer cells. HR-MALDI IMS firstly demonstrated the possible association between the PI profiles of breast cancer cells and their invasion/metastasis ability. Although this is a preliminary study, HR-MALDI IMS could be a useful modality to visualize the reprogramming of the lipid metabolism which reflects the cancer cell phenotype. Citation Format: Masahiro Kawashima, Nobuko Kawaguchi-Sakita, Masahiro Sugimoto, Taka-Aki Sato, Koichi Tanaka, Masakazu Toi. High-resolution MALDI imaging mass spectrometry demonstrated the correlation between phosphatidylinositol profiles and invasion/metastasis in human breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4959. doi:10.1158/1538-7445.AM2014-4959

Cholesterol and F‐actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane

Synaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-β-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.

The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum: a comparative approach

Dehydration leads to different physiological and biochemical responses in plants. We analysed the lipid composition and the expression of genes involved in lipid biosynthesis in the desiccation-tolerant plant Craterostigma plantagineum. A comparative approach was carried out with Lindernia brevidens (desiccation tolerant) and two desiccation-sensitive species, Lindernia subracemosa and Arabidopsis thaliana. In C.plantagineum the total lipid content remained constant while the lipid composition underwent major changes during desiccation. The most prominent change was the removal of monogalactosyldiacylglycerol (MGDG) from the thylakoids. Analysis of molecular species composition revealed that around 50% of 36:x (number of carbons in the acyl chains: number of double bonds) MGDG was hydrolysed and diacylglycerol (DAG) used for phospholipid synthesis, while another MGDG fraction was converted into digalactosyldiacylglycerol via the DGD1/DGD2 pathway and subsequently into oligogalactolipids by SFR2. 36:x-DAG was also employed for the synthesis of triacylglycerol. Phosphatidic acid (PA) increased in C.plantagineum, L.brevidens, and L.subracemosa, in agreement with a role of PA as an intermediate of lipid turnover and of phospholipase D in signalling during desiccation. 34:x-DAG, presumably derived from de novo assembly, was converted into phosphatidylinositol (PI) in C.plantagineum and L.brevidens, but not in desiccation-sensitive plants, suggesting that PI is involved in acquisition of desiccation tolerance. The accumulation of oligogalactolipids and PI in the chloroplast and extraplastidial membranes, respectively, increases the concentration of hydroxyl groups and enhances the ratio of bilayer- to non-bilayer-forming lipids, thus contributing to protein and membrane stabilization.

SH2‐Containing Inositol 5′‐Phosphatase 2 Selectively Impairs Hypothalamic Insulin Signalling and Regulation of Food Intake in Mice

SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a lipid phosphatase that negatively regulates the metabolic signalling of insulin in peripheral tissues; however, the expression of SHIP2 in the hypothalamus and its functional roles are largely unknown. In the present study, immunohistochemical analysis demonstrated that SHIP2 protein exists in neuronal cells expressing neuropeptide Y and pro-opiomelanocortin in the arcuate nucleus of the hypothalamus in C57BL/6J mice. Interestingly, the expression levels of SHIP2 in the hypothalamus were elevated in aged C57BL/6J mice and diabetic db/db mice. To clarify the significance of the increased expression of SHIP2 in the hypothalamus, we examined the central effects of insulin and leptin in transgenic mice overexpressing SHIP2 (SHIP2-Tg). Accumulation of phosphatidylinositol (3,4,5)-trisphosphate and phosphorylation of Akt in the hypothalamus, induced by i.c.v. injection of insulin, were attenuated in SHIP2-Tg compared to wild-type mice, whereas leptin-induced phosphorylation of signal transducer and activator of transcription 3 in the hypothalamus was comparable between them. The suppression of food intake after i.c.v. administration of insulin (but not leptin) was attenuated consistently in SHIP2-Tg. In addition, SHIP2-Tg showed increased food consumption after starvation and become heavier with visceral fat accumulation than wild-type mice, despite normal levels of oxygen consumption and spontaneous movement. These results suggest that SHIP2 contributes to the regulation of food intake mainly via the attenuation of insulin signalling in the hypothalamus of mice.

The mTOR (Mammalian Target of Rapamycin) Kinase Maintains Integrity of mTOR Complex 2

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.

Chronic alteration in phosphatidylinositol 4,5-biphosphate levels regulates capsaicin and mustard oil responses

There is an agreement that acute (in minutes) hydrolysis and accumulation of phosphatidylinositol 4,5-bisphosphate (PIP(2) ) modulate TRPV1 and TRPA1 activities. Because inflammation results in PIP(2) depletion, persisting for long periods (hours to days) in pain models and in the clinic, we examined whether chronic depletion and accumulation of PIP(2) affect capsaicin (CAP) and mustard oil (MO) responses. In addition, we wanted to evaluate whether the effects of PIP(2) depend on TRPV1 and TRPA1 coexpression and whether the PIP(2) actions vary in expression cells vs. sensory neurons. Chronic PIP(2) production was stimulated by overexpression of phosphatidylinositol-4-phosphate-5-kinase, and PIP(2) -specific phospholipid 5'-phosphatase was selected to reduce plasma membrane levels of PIP(2) . Our results demonstrate that CAP (100 nM) responses and receptor tachyphylaxis are not significantly influenced by chronic changes in PIP(2) levels in wild-type (WT) or TRPA1 null-mutant sensory neurons as well as CHO cells expressing TRPV1 alone or with TRPA1. However, low concentrations of CAP (20 nM) produced a higher response after PIP(2) depletion in cells containing TRPV1 alone but not TRPV1 together with TRPA1. MO (25 μM) responses were also not affected by PIP(2) in WT sensory neurons and cells coexpressing TRPA1 and TRPV1. In contrast, PIP(2) reduction leads to pronounced tachyphylaxis to MO in cells with both channels. Chronic effect of PIP(2) on TRPA1 activity depends on presence of the TRPV1 channel and cell type (CHO vs. sensory neurons). In summary, chronic alterations in PIP(2) levels regulate magnitude of CAP and MO responses as well as MO tachyphylaxis. This regulation depends on coexpression profile of TRPA1 and TRPV1 and cell type.

Association of HLA Haplotypes with Paroxysmal Nocturnal Hemoglobinuria

The pathologies of paroxysmal nocturnal hemoglobinuria (PNH) are primarily caused by somatic mutation in the PIG-A gene in hematopoietic stem cells resulting in glycosyl phosphatidylinositol deficiency and accumulation of phosphatidylinositol (PI) in plasma membranes. The mechanism of pathologic clone domination over normal hematopoietic clones in PNH patients is not yet understood. Forty-four PNH patients, including 9 with aplastic anemia traits (AA/PNH), 31 without full aplasia in bone marrow (de novo PNH, or dn/PNH), and 4 with unclassified PNH, and 200 ethnically matched controls were tested for the HLA A, B, C, DRB1, and DQB1 alleles and haplotype associations. The top block association analysis showed the primary association of PNH with 3 haplotype fragments: the class I fragment A*2501-Cw*1203-B*1801 (risk ratio [RR], 6.64; P = .00012), and 2 class II fragments: DRB1*1501-DQB1*0602 (RR, 7.09; P = .0000015) and DRB1*0401-DQB1*0301 (RR, 6.76, P = .0093). The stratified analysis revealed that the A*2501-Cw*1203-B*1801 haplotype associated preferentially with AA/PNH, and its component HLA molecule showed immunodominant antiapoptotic peptides derived from PI-activated phospholipase D; whereas the DRB1*1501-DQB1*0602 haplotype was associated strongly with dn/PNH and presented immunodominant class II–derived autopeptides. We concluded that certain HLA haplotypes were associated with PNH much more strongly than their allelic components. At least 3 HLA haplotype blocks ( A*2501-Cw*1203-B*1801, DRB1*1501-DQB1*0602, and DRB1*0401-DQB1*0301) were primarily associated with PNH. Our results supported the hypothesis of the roles in AA/PNH of antiapoptotic and in dn/PNH of autoimmune mechanisms.

Molecular mechanism of membrane targeting by the GRP1 PH domain*s⃞

The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P(3) with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P(3) binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P(3) triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P(3) binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P(3) vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns(3,4,5)P(3) and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P(3)-containing vesicles is further amplified (by approximately 6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.

Molecular Mechanism of Action of Monocyclam Versus Bicyclam Non-peptide Antagonists in the CXCR4 Chemokine Receptor

AMD3465 is a novel, nonpeptide CXCR4 antagonist and a potent inhibitor of HIV cell entry in that one of the four-nitrogen cyclam rings of the symmetrical, prototype bicyclam antagonist AMD3100 has been replaced by a two-nitrogen N-pyridinylmethylene moiety. This substitution induced an 8-fold higher affinity as determined against (125)I-12G5 monoclonal CXCR4 antibody binding, and a 22-fold higher potency in inhibition of CXCL12-induced signaling through phosphatidylinositol accumulation. Mutational mapping of AMD3465 and a series of analogs of this in a library of 23 mutants covering the main ligand binding pocket of the CXCR4 receptor demonstrated that the single cyclam ring of AMD3465 binds in the pocket around AspIV:20 (Asp(171)), in analogy with AMD3100, whereas the N-pyridinylmethylene moiety mimics the other cyclam ring through interactions with the two acidic anchor-point residues in transmembrane (TM)-VI (AspVI:23/Asp(262)) and TM-VII (GluVII:06/Glu(288)). Importantly, AMD3465 has picked up novel interaction sites, for example, His(281) located at the interface of extracellular loop 3 and TM-VII and HisIII:05 (His(113)) in the middle of the binding pocket. It is concluded that the simple N-pyridinylmethylene moiety of AMD3465 substitutes for one of the complex cyclam moieties of AMD3100 through an improved and in fact expanded interaction pattern mainly with residues located in the extracellular segments of TM-VI and -VII of the CXCR4 receptor. It is suggested that the remaining cyclam ring of AMD3465, which ensures the efficacious blocking of the receptor, in a similar manner can be replaced by chemical moieties allowing for, for example, oral bioavailability.

Involvement of the Phosphatidylinositol 3-Kinase/Rac1 and Cdc42 Pathways in Radial Migration of Cortical Neurons

During cortical development, newly generated neurons migrate radially toward their final positions. Although several candidate genes essential for this radial migration have been reported, the signaling pathways regulating it are largely unclear. Here we studied the role of phosphatidylinositol (PI) 3-kinase and its downstream signaling molecules in the radial migration of cortical neurons in vivo and in vitro. The expression of constitutively active and dominant-negative PI 3-kinases markedly inhibited radial migration. In the neocortical slice culture, a PI 3-kinase inhibitor suppressed the formation of GTP-bound Rac1 and Cdc42 and radial migration. Constitutively active and dominant-negative forms of Rac1 and Cdc42 but not Akt also significantly inhibited radial migration. In migrating neurons, wild-type Rac1 and Cdc42 showed different localizations; Rac1 localized to the plasma membrane and Cdc42 to the perinuclear region on the side of the leading processes. These results suggest that both the PI 3-kinase/Rac1 and Cdc42 pathways are involved in the radial migration of cortical neurons and that they have different roles.

Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes.

In ascidians, as in mammals, sperm trigger repetitive Ca2+-waves that originate from cortical pacemakers situated in the vegetal hemisphere of the zygotes. In ascidians, a vegetal protrusion termed the contraction pole (CP) acts as the Ca2+-wave pacemaker, but the mechanism that underlies the generation of a Ca2+-wave pacemaker is not known. Here, we tested four hypotheses to determine which factors at the CP are involved in setting the pace of the ascidian Ca2+-wave pacemaker: (1) localized Ca2+ influx; (2) accumulation of phosphatidylinositol (4,5)bisphosphate [PtdIns(4,5)P2]; (3) accumulation of cortical endoplasmic reticulum (cER); and (4) enrichment of the sperm activating factor. We developed a method of dynamically monitoring the location of the CP during fertilization using a plekstrin homology (PH) domain from phospholipase Cdelta1 coupled to green fluorescent protein (GFP) that binds PtdIns(4,5)P2. We found that eggs in Ca2+-free sea water displayed Ca2+ waves that originated from the CP, showing that enhanced CP Ca2+ influx does not determine the origin of the pacemaker. Also, disruption of the PH::GFP-labelled CP once it had formed did not dislodge the Ca2+-wave pacemaker from that site. Next, when we prevented the accumulation of cER at the CP, all of the Ca2+ waves came from the site of sperm-egg fusion and the frequency of Ca2+ oscillations was unaltered. These data show that local Ca2+ influx, the accumulation of PtdIns(4,5)P2 and cER at the CP are not required for Ca2+-wave pacemaker function and instead suggest that a factor associated with the sperm determines the site of the Ca2+-wave pacemaker. Finally, when we injected ascidian sperm extract into the centre of unfertilized ascidian eggs that had been treated with microfilament- and microtubule-disrupting drugs, all the Ca2+ waves still originated from near the plasma membrane, showing that the sperm factor does not require an intact cortex if it is enriched near the plasma membrane (PM). We suggest that the Ca2+-releasing sperm factor might be tethered near or on the PM and that following the cortical contraction, it is translocated to the vegetal CP, thus making that site act as a Ca2+-wave pacemaker.

Competition at the Leading Edge

Dictyostelium discoideum amoebae respond to the chemoattractant adenosine 3′,5′-monophosphate (cAMP) by aggregating to form a multicellular organism. By activating a heterotrimeric GTP-binding protein (G protein)-coupled receptor, cAMP initiates a signaling cascade that coordinately regulates phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphatase. The result is localized accumulation of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) at the cell's leading edge. Proteins harboring a pleckstrin homology (PH) domain that bind to PIP 3 translocate to this region, propagating further downstream signaling that leads to chemotaxis. Luo et al . report that the inositol pyrophosphate InsP 7 competes with PIP 3 for binding PH domain-containing proteins. Exposure of D. discoideum cells to cAMP caused a rapid increase in InsP 7 . Deleting the InsP 6 kinase, the enzyme that generates InsP 7 , resulted in a more rapid aggregation response, indicating increased sensitivity to cAMP. Increased translocation of a fluorescently labeled PH domain to the leading edge of chemotaxing cells corresponded with this increased sensitivity. No increase in PIP 3 was detected. InsP 7 also associated with PH domains in vitro and in vivo and inhibited PH domain interaction with PIP 3 . The study suggests that InsP 7 controls the translocation of PH domain-containing proteins and thus can suppress chemotactic responsiveness. H. R. Luo, Y. E. Huang, J. C. Chen, A. Saiardi, M. Iijima, K. Ye, Y. Huang, E. Nagata, P. Devreotes, S. H. Solomon, Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions. Cell 114 , 559-572 (2003). [Online Journal]

Homeobox gene Cdx1 regulates Ras, Rho and PI3 kinase pathways leading to transformation and tumorigenesis of intestinal epithelial cells.

The Cdx1 homeobox gene encodes for an intestine-specific transcription factor involved in the control of proliferation and differentiation of epithelial cells. Although it has been indicated that Cdx1 may act as a proto-oncogene in cultured fibroblasts, its direct role in the regulation of intestinal tumorigenesis has not been demonstrated. Here we show that expression of Cdx1 in an intestinal epithelial cell line (IEC-6) induces anchorage-independent growth in soft agar and promotes the formation of adenocarcinoma in vivo. The phenotype of Cdx1-induced tumors was exacerbated when IEC-6/Cdx1 cells were injected together with matrigel containing mitogens and extracellular matrix components. These changes were correlated with an increase in the GTP-bound form of Ras, modulation of Cdc42 and Rho-A activities, and accumulation of phosphatidyl inositol 3 (PI3) kinase products. Moreover, combined inhibition of Ras/Rho and PI3 kinase signaling by synthethic inhibitors blocked colony formation of IEC-6/Cdx1 cells in soft agar. Taken together, these results demonstrate a direct involvement of Cdx1, and its collaboration with Ras, Rho and PI3 kinase pathways, in transformation and tumorigenesis of intestinal epithelial cells.