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Peter J Sims - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of the Tyrosine Phosphorylation of Phospholipid Scramblase 1 in Mast Cells That Are Stimulated through the High-Affinity IgE Receptor
    2016
    Co-Authors: Asma Kassas, Therese Wiedmer, Peter J Sims, Renato C Monteiro, Ulrich Blank, Ivan C Moura, Yumi Yamashita, Jorg Scheffel, Juan Rivera, Nicolas Charles
    Abstract:

    Engagement of high-affinity immunoglobulin E receptors (FceRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FceRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRc chain. FceRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FceRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FceRI-activated mast cells and that PLSCR

  • regulation of the tyrosine phosphorylation of Phospholipid Scramblase 1 in mast cells that are stimulated through the high affinity ige receptor
    PLOS ONE, 2014
    Co-Authors: Asma Kassas, Therese Wiedmer, Peter J Sims, Renato C Monteiro, Ulrich Blank, Ivan C Moura, Yumi Yamashita, Jorg Scheffel, Claudine Guerinmarchand, Juan A Rivera
    Abstract:

    Engagement of high-affinity immunoglobulin E receptors (FceRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FceRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRγ chain. FceRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FceRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FceRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.

  • Phospholipid Scramblase 1 an interferon regulated gene located at 3q23 is regulated by snon skil in ovarian cancer cells
    Molecular Cancer, 2013
    Co-Authors: Karthik M Kodigepalli, Peter J Sims, Pavana Anur, Paul T Spellman, Meera Nanjundan
    Abstract:

    Treatment of advanced stage ovarian cancer continues to be challenging due to acquired drug resistance and lack of early stage biomarkers. Genes identified to be aberrantly expressed at the 3q26.2 locus (i.e. SnoN/SkiL) have been implicated in ovarian cancer pathophysiology. We have previously shown that SnoN expression is increased in advanced stage ovarian cancers and alters cellular response to arsenic trioxide (As2O3). We now demonstrate increased DNA copy number levels (TCGA data) of Phospholipid Scramblase 1 (PLSCR1, located at 3q23) whose transcript expression in ovarian cell lines is highly correlated with SnoN mRNA. Interestingly, SnoN can modulate PLSCR1 mRNA levels in the absence/presence of interferon (IFN-2α). Both IFN-2α and As2O3 treatment can modulate PLSCR1 mRNA levels in ovarian carcinoma cells. However, SnoN siRNA does not lead to altered PLSCR1 protein implicating other events needed to modulate its protein levels. In addition, we report that PLSCR1 can modulate aspects of the As2O3 cellular response. Our findings warrant further investigation into the role of PLSCR1 in ovarian cancer development and chemoresistance.

  • abstract 265 stat 1 activation enhances Phospholipid Scramblase 1 mediated apoptosis in aromatase inhibitor resistant breast cancer cells
    Cancer Research, 2012
    Co-Authors: Charlene Brewer, Peter J Sims, Rifat Jan, Analese Smith, Tabitha King, Joan S Lewiswambi
    Abstract:

    The signal transducer and activator of transcription 1 (STAT-1) protein is essential for signaling by interferons (IFNs), which, in addition to their role in innate immunity, serve as potent inhibitors of growth and promoters of apoptosis. Likewise, the Phospholipid Scramblase 1 (PLSCR-1) protein is also induced by interferon and is responsible for the translocation of Phospholipids between the lipid bilayer of a cell membrane which occurs during apoptosis. While there is increasing evidence to suggest that STAT-1 and PLSCR-1 play a direct role in apoptosis, the molecular mechanism by which these proteins regulate apoptotic cell death remains unclear. Previously, our laboratory has reported the development of an estrogen receptor alpha (ERα)-positive breast cancer cell line, MCF-7:5C, which is resistant to long term estrogen deprivation (i.e resistant to aromatase inhibitors) but undergoes apoptosis in the presence of physiologic concentrations of 17β-estradiol (E2). Global gene expression profile of MCF-7:5C cells has previously revealed that estradiol-induced apoptosis is associated with significant upregulation of several proinflammatory genes including; IFNs, STAT1, and PLSCR-1. In the present study, we investigated the role of STAT-1 and PLSCR-1 in estradiol-induced apoptosis in MCF-7:5C breast cancer cells. We found that PLSCR-1 and STAT-1 proteins were constitutively overexpressed by ∼20- and 10-fold, respectively, in MCF-7:5C cells compared to hormone-responsive MCF-7 and T47D breast cancer cells and siRNA suppression of PLSCR-1 or STAT-1 expression in MCF-7:5C cells completely blocked their ability to undergo apoptosis in the presence of estradiol and/or interferon-alpha (IFNα) but not other apoptosis-inducing agents such as taxol and etoposide. Immunofluoresence analysis of MCF-7:5C cells indicated that PLSCR-1 and STAT-1 were overexpressed and localized primarily in the cytoplasm of these cells; however, in the presence of IFNα and E2, a significant portion of PLSCR-1 and STAT-1 translocated to the nucleus and was associated with apoptosis. We also found that increased STAT-1 and PLSCR1 activation inhibited the expression of prosurvival (BCL2, BCL-xL) and induced the expression of proapoptotic members (BAK, mitochondrial BAX) of the BCL2 family and was associated with increased mitochondrial membrane permeability and activation of CASP7, CASP8, and CASP9, as well as suppression of pAKT and NF-κB. Overall, our data demonstrate that STAT-1 and PLSCR-1 play a critical role in sensitizing aromatase inhibitor resistant breast cancer cells to estradiol-induced apoptosis and that IFNα combined with E2 might be an effective treatment option for patients with endocrine resistant disease. This work was supported by the NIH Career Development Grant K01CA120051 01A2 and the Hollenbach Foundation Grant (JSLW). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 265. doi:1538-7445.AM2012-265

  • a minimal nuclear localization signal nls in human Phospholipid Scramblase 4 that binds only the minor nls binding site of importin α1
    Journal of Biological Chemistry, 2011
    Co-Authors: Kaylen Lott, Peter J Sims, Anshul Bhardwaj, Gino Cingolani
    Abstract:

    Abstract Importin α1 can bind classical nuclear localization signals (NLSs) in two NLS-binding sites, known as “major” and “minor.” The major site is located between ARM repeats 2–4, whereas the minor site spans ARM 7–8. In this study, we have characterized the cellular localization of human Phospholipid Scramblase 4 (hPLSCR4), a member of the Phospholipid Scramblase protein family. We identified a minimal NLS in hPLSCR4 (273GSIIRKWN280) that contains only two basic amino acids. This NLS is both necessary for nuclear localization of hPLSCR4 in transfected HeLa cells and sufficient for nuclear import of a non-diffusible cargo in permeabilized cells. Mutation of only one of the two basic residues, Arg277, correlates with loss of nuclear localization, suggesting this amino acid plays a key role in nuclear transport. Crystallographic analysis of mammalian importin α1 in complex with the hPLSCR4-NLS reveals this minimal NLS binds specifically and exclusively to the minor binding site of importin α. These data provide the first structural and functional evidence of a novel NLS-binding mode in importin α1 that uses only the minor groove as the exclusive site for nuclear import of nonclassical cargos.

Therese Wiedmer - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of the Tyrosine Phosphorylation of Phospholipid Scramblase 1 in Mast Cells That Are Stimulated through the High-Affinity IgE Receptor
    2016
    Co-Authors: Asma Kassas, Therese Wiedmer, Peter J Sims, Renato C Monteiro, Ulrich Blank, Ivan C Moura, Yumi Yamashita, Jorg Scheffel, Juan Rivera, Nicolas Charles
    Abstract:

    Engagement of high-affinity immunoglobulin E receptors (FceRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FceRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRc chain. FceRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FceRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FceRI-activated mast cells and that PLSCR

  • regulation of the tyrosine phosphorylation of Phospholipid Scramblase 1 in mast cells that are stimulated through the high affinity ige receptor
    PLOS ONE, 2014
    Co-Authors: Asma Kassas, Therese Wiedmer, Peter J Sims, Renato C Monteiro, Ulrich Blank, Ivan C Moura, Yumi Yamashita, Jorg Scheffel, Claudine Guerinmarchand, Juan A Rivera
    Abstract:

    Engagement of high-affinity immunoglobulin E receptors (FceRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FceRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRγ chain. FceRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FceRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FceRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.

  • expression of the Phospholipid Scramblase plscr gene family during the acute phase response
    Biochimica et Biophysica Acta, 2007
    Co-Authors: Peter J Sims, Therese Wiedmer, Arthur H Moser, Judy K Shigenaga, Carl Grunfeld, Kenneth R Feingold
    Abstract:

    Phospholipid Scramblase 1 (PLSCR1) is a member of PLSCR gene family that has been implicated in multiple cellular processes including movement of Phospholipids, gene regulation, immuno-activation, and cell proliferation/apoptosis. In the present study, we identified PLSCR1 as a positive intracellular acute phase protein that is upregulated by LPS in liver, heart, and adipose tissue, but not skeletal muscle. LPS administration resulted in a marked increase in PLSCR1 mRNA and protein levels in the liver. This stimulation occurred rapidly (within 2 h), and was very sensitive to LPS (half-maximal response at 0.1 microg/mouse). Moreover, two other APR-inducers, zymosan and turpentine, also produced significant increases in PLSCR1 mRNA and protein levels, indicating that PLSCR1 was stimulated in a number of models of the APR. To determine signaling pathways by which LPS stimulated PLSCR1, we examined the effect of proinflammatory cytokines in vitro and in vivo. TNFalpha, IL-1beta, and IL-6 all stimulated PLSCR1 in cultured Hep B3 hepatocytes, whereas only TNFalpha stimulated PLSCR1 in cultured 3T3-L1 adipocytes, suggesting cell type-specific effects of cytokines. Furthermore, the LPS-stimulated increase in liver PLSCR1 mRNA was greatly attenuated by 80% in TNFalpha and IL-1beta receptor null mice as compared to wild-type controls. In contrast, PLSCR1 levels in adipose tissue were induced to a similar extent in TNFalpha and IL-1beta receptor null mice and controls. These results indicate that maximal stimulation of PLSCR1 by LPS in liver required TNFalpha and/or IL-1beta, whereas the stimulation of PLSCR1 in adipose tissue is not dependent on TNFalpha and/or IL-1beta. These data provide evidence that PLSCR1 is a positive intracellular acute phase protein with a tissue-specific mechanism for up-regulation.

  • antileukemic roles of human Phospholipid Scramblase 1 gene evidence from inducible plscr1 expressing leukemic cells
    Oncogene, 2006
    Co-Authors: Yuji Huang, Therese Wiedmer, Peter J Sims, Guoqiang Chen, Qiwu Zhao, C X Zhou, K W Zhao
    Abstract:

    Phospholipid Scramblase 1 (PLSCR1) is a multiply palmitoylated protein which is localized in either the cell membrane or nucleus depending on its palmitoylated state. The increasing evidence showed the biological roles of PLSCR1 in cell signaling, maturation and apoptosis. To investigate the functions of PLSCR1 in leukemic cells, we generated an inducible PLSCR1-expressing cell line using myeloid leukemic U937 cells. In this cell line, PLSCR1 was tightly regulated and induced upon tetracycline withdrawal. Our results showed that inducible PLSCR1 expression arrested the proliferation of U937 cells at G1 phase. Meanwhile, PLSCR1-overexpressing U937 cells also underwent granulocyte-like differentiation with increased sensitivity to etoposide-induced apoptosis. Furthermore, we also found that PLSCR1 induction increased cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 proteins, together with downregulation of S phase kinase-associated protein 2 (SKP2), an F-box subunit of the ubiquitin-ligase complex that targets proteins for degradation. Additionally, PLSCR1 induction significantly decreased c-Myc protein and antiapoptotic Bcl-2 protein. Although the exact mechanism by which PLSCR1 regulates these cellular events and gene expression remains unresolved, our results suggest that PLSCR1 plays the antagonistic role regarding leukemia development. These data will shed new insights into understanding the biochemical and biological functions of PLSCR1 protein.

  • Phospholipid Scramblase 1 binds to the promoter region of the inositol 1 4 5 triphosphate receptor type 1 gene to enhance its expression
    Journal of Biological Chemistry, 2005
    Co-Authors: Quansheng Zhou, Therese Wiedmer, Iris Benefraim, Jolawrence Bigcas, Daniela Junqueira, Peter J Sims
    Abstract:

    Phospholipid Scramblase 1 (PLSCR1) is a multiply palmitoylated, endofacial membrane protein originally identified based on its capacity to promote accelerated transbilayer Phospholipid movement in response to Ca(2+). Recent evidence suggests that this protein also participates in cell response to various growth factors and cytokines, influencing myeloid differentiation, tumor growth, and the antiviral activity of interferon. Whereas plasma membrane PLSCR1 was shown to be required for normal recruitment and activation of Src kinase by stimulated cell surface growth factor receptors, PLSCR1 was also found to traffic into the nucleus and to tightly bind to genomic DNA, suggesting a possible additional nuclear function. We now report evidence that PLSCR1 directly binds to the 5'-promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene (IP3R1) to enhance expression of the receptor. Probing a CpG island genomic library with PLSCR1 as bait identified four clones with avidity for PLSCR1, including a 191-bp fragment of the IP3R1 promoter. Using electrophoretic mobility shift and transcription reporter assays, the PLSCR1-binding site in IP3R1 was mapped to residues (-101)GTAACCATGTGGA(-89), and the segment spanning Met(86)-Glu(118) in PLSCR1 was identified to mediate its transcriptional activity. The significance of this interaction between PLSCR1 and IP3R1 in situ was confirmed by comparing levels of IP3R1 mRNA and protein in matched cells that either expressed or were deficient in PLSCR1. These data suggest that in addition to its role at the plasma membrane, effects of PLSCR1 on cell proliferative and maturational responses may also relate to alterations in expression of cellular IP3 receptors.

Sathyanarayana N Gummadi - One of the best experts on this subject based on the ideXlab platform.

  • Are cysteine residues of human Phospholipid Scramblase 1 essential for Pb^2+ and Hg^2+ binding-induced scrambling of Phospholipids?
    European Biophysics Journal, 2021
    Co-Authors: Ashok Kumar Shettihalli, Santosh Kumar Palanirajan, Sathyanarayana N Gummadi
    Abstract:

    Lead and mercury being common environmental pollutants are often associated with erythrocytes, where phosphatidylserine (PS) exposure-mediated procoagulant activation is induced. Human Phospholipid Scramblase 1 (hPLSCR1) identified in the erythrocyte membrane is a type II transmembrane protein involved in Ca^2+-dependent bidirectional scrambling of Phospholipids (PL) during blood coagulation, cell activation, and apoptosis. The prominent role of hPLSCR1 in Pb^2+ and Hg^2+ poisoning was demonstrated by a biochemical assay, where recombinant hPLSCR1 induced PL scrambling across bilayer with a higher binding affinity ( K _d) towards Hg^2+ (4.1 µM) and Pb^2+ (5.8 µM) than Ca^2+ (25.6 mM). The increased affinity could be the outcome of heavy metals interacting at auxiliary sites other than the calcium-binding motif of hPLSCR1. Similar to other metal-binding proteins, cysteine-based metal-binding motifs could be the potential additional binding sites in hPLSCR1. To explore the hypothesis, the cysteines were chemically modified, which significantly reduced only the Hg^2+- and Pb^2+-induced scrambling activity leaving Ca^2+-induced activity unaltered. Recombinant constructs with deletion of prominent cysteine residues and point mutation in the calcium-binding motif including Δ100-hPLSCR1, Δ160-hPLSCR1, and D275A-hPLSCR1 were generated, purified, and assayed for Scramblase activity. The cysteine-deleted constructs of hPLSCR1 showed reduced binding affinity ( K _d) for Hg^2+ and Pb^2+ without altering the Ca^2+-binding affinity whereas the point mutant had completely lost its affinity for Ca^2+ and reduced affinities for Hg^2+ and Pb^2+. The results accentuated the significance of cysteine residues as additional binding sites for heavy metal ions in hPLSCR1.

  • in vitro reconstitution and biochemical characterization of human Phospholipid Scramblase 3 Phospholipid specificity and metal ion binding studies
    Biological Chemistry, 2018
    Co-Authors: Santosh Kumar Palanirajan, Ulaganathan Sivagnanam, Sowmiya Murugan, Sathyanarayana N Gummadi
    Abstract:

    Human Phospholipid Scramblase 3 (hPLSCR3) is a single pass transmembrane protein that plays a vital role in fat metabolism, mitochondrial function, structure, maintenance and apoptosis. The mechanism of action of Scramblases remains still unknown, and the role of Scramblases in Phospholipid translocation is heavily debated. hPLSCR3 is the only member of Scramblase family localized to mitochondria and is involved in cardiolipin translocation at the mitochondrial membrane. Direct biochemical evidence of Phospholipid translocation by hPLSCR3 is yet to be reported. Functional assay in synthetic proteoliposomes upon Ca2+ and Mg2+ revealed that, apart from cardiolipin, recombinant hPLSCR3 translocates aminoPhospholipids such as NBD-PE and NBD-PS but not neutral Phospholipids. Point mutation in hPLSCR3 (F258V) resulted in decreased Ca2+ binding affinity. Functional assay with F258V-hPLSCR3 led to ~50% loss in Scramblase activity in the presence of Ca2+ and Mg2+. Metal ion-induced conformational changes were monitored by intrinsic tryptophan fluorescence, circular dichroism, surface hydrophobicity changes and aggregation studies. Our results revealed that Ca2+ and Mg2+ bind to hPLSCR3 and trigger conformational changes mediated by aggregation. In summary, we suggest that the metal ion-induced conformational change and the aggregation of the protein are essential for the Phospholipid translocation by hPLSCR3.

  • identification and characterization of the novel nuclease activity of human Phospholipid Scramblase 1
    BMC Biochemistry, 2016
    Co-Authors: Ulaganathan Sivagnanam, Shweta Narayana Murthy, Sathyanarayana N Gummadi
    Abstract:

    Background Human Phospholipid Scramblase 1 (hPLSCR1) was initially identified as a Ca2+ dependent Phospholipid translocator involved in disrupting membrane asymmetry. Recent reports revealed that hPLSCR1 acts as a multifunctional signaling molecule rather than functioning as Scramblase. hPLSCR1 is overexpressed in a variety of tumor cells and is known to interact with a number of protein molecules implying diverse functions.

  • two c myc binding sites are crucial in upregulating the expression of human Phospholipid Scramblase 1 gene
    Biochemical and Biophysical Research Communications, 2016
    Co-Authors: Janaki Manoja Vinnakota, Sathyanarayana N Gummadi
    Abstract:

    Abstract Human Phospholipid Scramblase 1 (hPLSCR1) is a type II endofacial membrane protein which mediates bi-directional transport of Phospholipids across the plasma membrane. hPLSCR1, a multifunctional protein with variety of roles in apoptosis, tumor progression, cell signaling and anti-viral defense. The expression of such a multifunctional protein should be under tight regulation. Apart from a single report showing snail mediated down regulation of hPLSCR1, the molecular mechanisms regulating the expression of Scramblases are not well elucidated. In this study we identified c-Myc as a transcriptional regulator of hPLSCR1. Transcription factor prediction tool ConSite predicted three binding sites for c-Myc. Reporter gene assays and western blot analysis revealed c-Myc mediated up regulation of hPLSCR1 expression. Deletion construct −790 lacking one c-Myc binding site showed a 27% decrease in promoter activity while deletion construct −469 lacking two c-Myc binding sites showed a 62% decrease in promoter activity. Site directed mutagenesis revealed the importance of c-Myc binding sites from −751 to −756 and −548 to −553 on the promoter of hPLSCR1in transcriptionally regulating the expression of hPLSCR1. The results were further confirmed by shRNA mediated knock down of endogenous c-Myc and in vivo interactions by ChIP assay.

  • the single c terminal helix of human Phospholipid Scramblase 1 is required for membrane insertion and scrambling activity
    FEBS Journal, 2013
    Co-Authors: Vincent Gerard Francis, Abdul M Mohammed, Gopala Krishna Aradhyam, Sathyanarayana N Gummadi
    Abstract:

    Human Phospholipid Scramblase 1 (hPLSCR1) belongs to the ATP-independent class of Phospholipid translocators which possess a single EF-hand-like Ca2+-binding motif and also a C-terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C-terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca2+ for ∆CTH-hPLSCR1 (Ka = 115 μm) compared with hPLSCR1 (Ka = 249 μm). Far UV-CD studies revealed that the CTH peptide adopts α-helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide-quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca2+ coordination and also plays an important role in the functional conformation of hPLSCR1.

Ray M Lee - One of the best experts on this subject based on the ideXlab platform.

  • role of Phospholipid Scramblase 3 in the regulation of tumor necrosis factor α induced apoptosis
    Biochemistry, 2008
    Co-Authors: Jihua Liu, Douglas Grossman, David Durrant, Raquel F Epand, Naiwen Chi, Ray M Lee
    Abstract:

    In tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis, tBid is targeted to mitochondria and causes cytochrome c release. We investigated the regulation of tBid-induced cytochrome c release and apoptosis by Phospholipid Scramblase 3 (PLS3). Overexpression of PLS3 enhanced, whereas downregulation of PLS3 delayed, TNF-alpha-induced apoptosis and targeting of tBid to mitochondria. On the basis of the theory that tBid targets mitochondrial cardiolipin, we hypothesize that PLS3 enhances translocation of cardiolipin to the mitochondrial surface to facilitate tBid targeting. NAO, a cardiolipin binding dye, was first used to quantify the distribution of cardiolipin. Overexpression of PLS3 increases, whereas downregulation of PLS3 decreases, the percentage of cardiolipin on the mitochondrial surface. Determination of the tBid binding capacity on the mitochondrial surface by FITC-labeled tBid(G94E) also confirmed that tBid binding capacity increased upon PLS3 overexpression and decreased with downregulation of PLS3. PLS3 activity, determined by a lipid flip-flop assay, was activated by calcium and tBid but inhibited by Bcl-2. Mutation of the calcium binding motif abolishes the lipid flip-flop activity of PLS3. PLS3 and tBid may form a bidirectional positive feedback loop that is antagonized by Bcl-2. Overexpression of PLS3 does not affect mitochondrial potential but does interfere with mitochondrial respiration and production of reactive oxygen species. These studies thus establish PLS3 as an important downstream effector of Bcl-2 and tBid in apoptosis.

  • phosphorylation of mitochondrial Phospholipid Scramblase 3 by protein kinase c δ induces its activation and facilitates mitochondrial targeting of tbid
    Journal of Cellular Biochemistry, 2007
    Co-Authors: Jihua Liu, Douglas Grossman, David Durrant, Trevor W Sweatman, Leonard Lothstein, Raquel F Epand, Ray M Lee
    Abstract:

    Phospholipid Scramblase 3 (PLS3) is a member of the Phospholipid Scramblase family present in mitochondria. PLS3 plays an important role in regulation of mitochondrial morphology, respiratory function, and apoptotic responses. PLS3 is phosphorylated by PKC-delta at Thr21 and is the mitochondrial target of PKC-delta-induced apoptosis. Cells with overexpression of PLS3, but not the phosphoinhibitory mutant PLS3(T21A), are more susceptible to apoptosis induced by AD198, an extranuclear targeted anthracycline that activates PKC-delta. Here we report that the phosphomimetic mutant of PLS3(T21D) by itself can induce apoptosis in HeLa cells. Using proteoliposomes with addition of pyrene-labeled phosphatidylcholine (PC) at the outer leaflet, we measured the lipid flip-flop activity of PLS3 and its phosphorylation mutant. PLS3(T21D) is more potent than wild-type PLS3 or PLS3(T21A) to transfer pyrene-PC from the outer leaflet to the inner leaflet of liposomes. Based on our previous finding that PLS3 enhances tBid-induced mitochondrial damages, we tested the hypothesis that PLS3 enhances cardiolipin translocation to mitochondrial surface and facilitates tBid targeting. Fluorescein-labeled tBid(G94E) was used as a probe to quantify cardiolipin on the surface of mitochondria. Mitochondria from cells treated with AD198 or cells expressing PLS3(T21D) had a higher level of tBid-binding capacity than control cells or cells expressing wild-type PLS3. These findings indicate that phosphorylation of PLS3 by PKC-delta induces PLS3 activation to facilitate mitochondrial targeting of tBid and apoptosis.

  • Phospholipid Scramblase 3 regulates cardiolipin de novo biosynthesis and its resynthesis in growing hela cells
    Biochemical Journal, 2007
    Co-Authors: Quyen Van, Jihua Liu, Kenneth R Feingold, Yuguang Shi, Ray M Lee, Grant M Hatch
    Abstract:

    PLS3 (Phospholipid Scramblase-3) is a new member of the family of Phospholipid Scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial Phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell ‘senses’ altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.

  • n benzyladriamycin 14 valerate ad198 induces apoptosis through protein kinase c δ induced phosphorylation of Phospholipid Scramblase 3
    Cancer Research, 2005
    Co-Authors: Jihua Liu, David Durrant, Trevor W Sweatman, Leonard Lothstein, Hung Sheng Yang, Ray M Lee
    Abstract:

    Phospholipid Scramblase 3 (PLS3) is an enzyme that plays a critical role in mitochondrial morphology, functions, and apoptotic response. During apoptosis, activated protein kinase C-δ (PKC-δ) translocates to mitochondria and phosphorylates PLS3. Here, we utilize an extranuclear-targeted anthracycline N -benzyladriamycin-14-valerate (AD198), a PKC-δ activator, to investigate the mechanism of PLS3 phosphorylation by PKC-δ. Overexpression of PLS3 enhanced, whereas down-regulation of PLS3 by small interfering RNA decreased, the sensitivity of AD198-induced apoptosis. Overexpression of PKC-δ, but not the kinase-defective PKC-δ, and AD198 treatment enhanced threonine phosphorylation of PLS3. The phosphorylated threonine was mapped to Thr 21 of PLS3. Mutation of Thr 21 to alanine did not affect mitochondrial localization of PLS3 but abolished threonine phosphorylation by PKC-δ in vitro and AD198-induced PLS3 phosphorylation in vivo . Expression of PLS3(T21A) in cells could not enhance AD198-induced apoptosis compared with expression of the wild-type PLS3. Using benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone and cyclosporine A, we also showed that AD198-induced PLS3 phosphorylation occurs upstream of caspase activation and independent of mitochondrial permeability transition. These studies establish that AD198-activated PKC-δ induces phosphorylation of mitochondrial PLS3 at Thr 21 and that PLS3 is a critical downstream effector of PKC-δ in AD198-induced apoptosis.

  • Phospholipid Scramblase 3 controls mitochondrial structure function and apoptotic response
    Molecular Cancer Research, 2003
    Co-Authors: Jihua Liu, Douglas Grossman, David Durrant, Jun Chen, Qiang Dai, Angela J Freeman, Tong Liu, Ray M Lee
    Abstract:

    Phospholipid Scramblase 3 (PLS3) is a newly recognized member of a family of proteins responsible for Phospholipid translocation between two lipid compartments. To study PLS3 function in mitochondria, we disrupted its conserved calcium-binding motif yielding an inactive mutant PLS3(F258V). Cells transfected with PLS3(F258V) exhibited reduced proliferative capacity. Mitochondrial analysis revealed that PLS3(F258V)-expressing cells have decreased mitochondrial mass shown by lower cytochrome c and cardiolipin (CL) content, poor mitochondrial respiration, and reduced oxygen consumption and intracellular ATP; whereas wild-type PLS3-transfected cells exhibit increased mitochondrial mass and enhanced respiration. Electron microscopic examination revealed that the mitochondria in PLS3(F258V)-expressing cells have densely packed cristae and are fewer in number and larger than those in control cells. The abnormal mitochondrial metabolism and structure in PLS3(F258V)-expressing cells were associated with decreased sensitivity to UV- and tBid-induced apoptosis and diminished translocation of CL to the mitochondrial outer membrane. In contrast, wild-type PLS3-transfected cells displayed increased sensitivity to apoptosis and enhanced CL translocation. These studies identify PLS3 as a critical regulator of mitochondrial structure and respiration, and CL transport in apoptosis.

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