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1.
Nat Commun ; 12(1): 6773, 2021 11 19.
Article in English | MEDLINE | ID: mdl-34799567

ABSTRACT

After inoculation by the bite of an infected mosquito, Plasmodium sporozoites enter the blood stream and infect the liver, where each infected cell produces thousands of merozoites. These in turn, infect red blood cells and cause malaria symptoms. To initiate a productive infection, sporozoites must exit the circulation by traversing the blood lining of the liver vessels after which they infect hepatocytes with unique specificity. We screened a phage display library for peptides that structurally mimic (mimotope) a sporozoite ligand for hepatocyte recognition. We identified HP1 (hepatocyte-binding peptide 1) that mimics a ~50 kDa sporozoite ligand (identified as phospholipid scramblase). Further, we show that HP1 interacts with a ~160 kDa hepatocyte membrane putative receptor (identified as carbamoyl-phosphate synthetase 1). Importantly, immunization of mice with the HP1 peptide partially protects them from infection by the rodent parasite P. berghei. Moreover, an antibody to the HP1 mimotope inhibits human parasite P. falciparum infection of human hepatocytes in culture. The sporozoite ligand for hepatocyte invasion is a potential novel pre-erythrocytic vaccine candidate.


Subject(s)
Malaria Vaccines/therapeutic use , Malaria, Falciparum/prevention & control , Phospholipid Transfer Proteins/immunology , Protozoan Proteins/immunology , Sporozoites/immunology , Animals , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Disease Models, Animal , Epitopes/immunology , Female , Hep G2 Cells , Hepatocytes/immunology , Hepatocytes/metabolism , Hepatocytes/parasitology , Humans , Liver/enzymology , Liver/parasitology , Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Male , Mice , Peptide Library , Phospholipid Transfer Proteins/isolation & purification , Phospholipid Transfer Proteins/metabolism , Plasmodium berghei/immunology , Plasmodium berghei/metabolism , Plasmodium falciparum/immunology , Plasmodium falciparum/metabolism , Primary Cell Culture , Protozoan Proteins/isolation & purification , Protozoan Proteins/metabolism , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sporozoites/metabolism , Vaccines, Subunit/immunology , Vaccines, Subunit/therapeutic use
2.
Mol Biochem Parasitol ; 243: 111374, 2021 05.
Article in English | MEDLINE | ID: mdl-33974939

ABSTRACT

Recent studies highlight the emerging role of lipids as important messengers in malaria parasite biology. In an attempt to identify interacting proteins and regulators of these dynamic and versatile molecules, we hypothesised the involvement of phospholipid translocases and their substrates in the infection of the host erythrocyte by the malaria parasite Plasmodium spp. Here, using a data base searching approach of the Plasmodium Genomics Resources (www.plasmodb.org), we have identified a putative phospholipid (PL) scramblase in P. falciparum (PfPLSCR) that is conserved across the genus and in closely related unicellular algae. By reconstituting recombinant PfPLSCR into liposomes, we demonstrate metal ion dependent PL translocase activity and substrate preference, confirming PfPLSCR as a bona fide scramblase. We show that PfPLSCR is expressed during asexual and sexual parasite development, localising to different membranous compartments of the parasite throughout the intra-erythrocytic life cycle. Two different gene knockout approaches, however, suggest that PfPLSCR is not essential for erythrocyte invasion and asexual parasite development, pointing towards a possible role in other stages of the parasite life cycle.


Subject(s)
Phospholipid Transfer Proteins/genetics , Plasmodium falciparum/enzymology , Protozoan Proteins/genetics , Amino Acid Sequence , Apicomplexa , Conserved Sequence , Erythrocytes/parasitology , Gene Expression Regulation, Enzymologic , Humans , Liposomes/chemistry , Liposomes/metabolism , Microorganisms, Genetically-Modified , Phospholipid Transfer Proteins/isolation & purification , Phospholipid Transfer Proteins/metabolism , Plasmodium falciparum/genetics , Plasmodium falciparum/pathogenicity , Protozoan Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
3.
Chem Res Toxicol ; 33(2): 553-564, 2020 02 17.
Article in English | MEDLINE | ID: mdl-31769662

ABSTRACT

Human phospholipid scramblases are a family of four homologous transmembrane proteins (hPLSCR1-4) mediating phospholipids (PLs) translocation in plasma membrane upon Ca2+ activation. hPLSCR3, the only homologue localized to mitochondria, plays a vital role in mitochondrial structure, function, maintenance, and apoptosis. Upon Ca2+ activation, hPLSCR3 mediates PL translocation at the mitochondrial membrane enhancing t-bid-induced cytochrome c release and apoptosis. Mitochondria are important target organelles for heavy-metals-induced apoptotic signaling cascade and are the central executioner of apoptosis to trigger. Pb2+ and Hg2+ toxicity mediates apoptosis by increased reactive oxygen species (ROS) and cytochrome c release from mitochondria. To discover the role of hPLSCR3 in heavy metal toxicity, hPLSCR3 was overexpressed as a recombinant protein in Escherichia coli Rosetta (DE3) and purified by affinity chromatography. The biochemical assay using synthetic proteoliposomes demonstrated that hPLSCR3 translocated aminophospholipids in the presence of micromolar concentrations of Pb2+ and Hg2+. A point mutation in the Ca2+-binding motif (F258V) led to a ∼60% loss in the functional activity and decreased binding affinities for Pb2+ and Hg2+ implying that the divalent heavy metal ions bind to the Ca2+-binding motif. This was further affirmed by the characteristic spectra observed with stains-all dye. The conformational changes upon heavy metal binding were monitored by circular dichroism, intrinsic tryptophan fluorescence, and light-scattering studies. Our results revealed that Pb2+ and Hg2+ bind to hPLSCR3 with higher affinity than Ca2+ thus mediating scramblase activity. To summarize, this is the first biochemical evidence for heavy metals binding to the mitochondrial membrane protein leading to bidirectional translocation of PLs specifically toward phosphatidylethanolamine.


Subject(s)
Apoptosis/drug effects , Lead/pharmacology , Mercury/pharmacology , Mitochondria/drug effects , Phospholipid Transfer Proteins/metabolism , Phospholipids/antagonists & inhibitors , Calcium/chemistry , Calcium/pharmacology , Dose-Response Relationship, Drug , Humans , Lead/chemistry , Mercury/chemistry , Mitochondria/metabolism , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/isolation & purification , Phospholipids/metabolism , Structure-Activity Relationship
4.
Methods Mol Biol ; 1949: 181-199, 2019.
Article in English | MEDLINE | ID: mdl-30790257

ABSTRACT

The distribution of different lipid species between the two leaflets is tightly regulated and underlies the concerted action of distinct catalytic entities. While flippases and floppases establish membrane asymmetry, scramblases randomize the lipid distribution and play pivotal roles during blood clotting, apoptosis, and in processes such as N-linked glycosylation of proteins. The recent discovery of TMEM16 family members acting as scramblases has led to an increasing demand for developing protocols tailored for TMEM16 proteins to enable functional investigations of their scrambling activity. Here we describe a protocol for the expression, purification, and functional reconstitution of TMEM16 proteins into preformed liposomes and measurement of their scrambling activity using fluorescence-labeled lipid derivatives. The reconstitution involves extrusion of liposomes through a membrane, destabilization of liposomes using Triton X-100, and stepwise detergent removal by adsorption on styryl-beads. The scrambling assay is based on the selective bleaching of nitrobenzoxadiazol fluorescent lipids on the outer leaflet of liposomes by the membrane-impermeant reducing agent sodium dithionite. The assay allows conclusions on the substrate specificity and on the kinetics of the transported lipids as shown with the example of a Ca2+-activated TMEM16 scramblase from the fungus Nectria haematococca (nhTMEM16).


Subject(s)
Anoctamins/metabolism , Phospholipid Transfer Proteins/metabolism , Proteolipids/metabolism , Anoctamins/isolation & purification , Phospholipid Transfer Proteins/isolation & purification , Proteolipids/chemistry
5.
Anal Biochem ; 556: 104-111, 2018 09 01.
Article in English | MEDLINE | ID: mdl-29964029

ABSTRACT

Human phospholipid scramblase 1 (hPLSCR1) is an ATP independent, Ca2+ dependent transmembrane protein mediating bidirectional translocation of phospholipids across the lipid bilayer but the mechanism of scrambling is unknown. Determination of the hPLSCR1 structure would help understand the mechanism and its multi-functional property. Recombinant hPLSCR1 forms inclusion bodies (IBs), when over-expressed in E. coli BL21 (DE3) and recovery of active hPLSCR1 from IBs, were time-consuming and resulted in low protein yield. This study aims to optimize and enhance the expression and purification of active recombinant hPLSCR1 by various strategies. Additives including stabilizers and detergents were added during cell lysis to improve the recovery of soluble hPLSCR1. Five E. coli strains, BL21 (DE3), C43 (DE3), Rosetta, BL21-CodonPlus-RP, and BL21 (DE3) pLysS were screened for maximum yield of soluble protein but localized in IBs. To recover hPLSCR1 from IBs, different additives were added of which, 0.3% N-lauroyl sarcosine (NLS) recovered ∼50% of bioactive hPLSCR1 from IBs. E. coli C43 (DE3) gave higher yields of purified protein (7.76 mg/g cell) followed by E. coli BL21 (DE3) pLysS (5.87 mg/g cell). This report describes a rapid and efficient method for solubilizing membrane proteins from inclusion bodies with a higher recovery.


Subject(s)
Inclusion Bodies/chemistry , Phospholipid Transfer Proteins , Escherichia coli/chemistry , Escherichia coli/metabolism , Humans , Phospholipid Transfer Proteins/biosynthesis , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/isolation & purification , Protein Stability , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
6.
Plant Cell Rep ; 35(8): 1671-86, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27061906

ABSTRACT

KEY MESSAGE: A Sec14-like protein, ZmSEC14p , from maize was structurally analyzed and functionally tested. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.


Subject(s)
Cold Temperature , Phospholipid Transfer Proteins/metabolism , Plant Proteins/metabolism , Zea mays/metabolism , Abscisic Acid/pharmacology , Adaptation, Physiological/drug effects , Adaptation, Physiological/genetics , Amino Acid Sequence , Antioxidants/metabolism , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/physiology , Freezing , Gene Expression Profiling , Gene Expression Regulation, Plant/drug effects , Genes, Plant , Germination/drug effects , Green Fluorescent Proteins/metabolism , Onions/cytology , Phosphoinositide Phospholipase C/metabolism , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/isolation & purification , Phylogeny , Plant Epidermis/cytology , Plant Epidermis/drug effects , Plant Leaves/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/isolation & purification , Plants, Genetically Modified , Proline/metabolism , Reactive Oxygen Species/metabolism , Seeds/drug effects , Seeds/growth & development , Sodium Chloride/pharmacology , Stress, Physiological/drug effects , Stress, Physiological/genetics , Subcellular Fractions/metabolism , Zea mays/drug effects , Zea mays/genetics , Zea mays/physiology
7.
Biochimie ; 107 Pt B: 223-34, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25223890

ABSTRACT

Caveolin is the protein marker of caveola-mediated endocytosis. Previously, we demonstrated by immunoblotting and immunofluorescence that an anti-chick embryo caveolin-1 monoclonal antibody (mAb) recognizes a protein in amoeba extracts. Nevertheless, the caveolin-1 gene is absent in the Entamoeba histolytica genome database. In this work, the goal was to isolate, identify and characterize the protein that cross-reacts with chick embryo caveolin-1. We identified the protein using a proteomic approach, and the complete gene was cloned and sequenced. The identified protein, E. histolytica phosphatidylcholine transfer protein-like (EhPCTP-L), is a member of the StAR-related lipid transfer (START) protein superfamily. The human homolog binds and transfers phosphatidylcholine (PC) and phosphatidylethanolamine (PE) between model membranes in vitro; however, the physiological role of PCTP-L remains elusive. Studies in silico showed that EhPCTP-L has a central START domain and also contains a C-terminal intrinsically disordered region. The anti-rEhPCTP-L antibody demonstrated that EhPCTP-L is found in the plasma membrane and cytosol, which is in agreement with previous reports on the human counterpart. This result points to the plasma membrane as one possible target membrane for EhPCTP-L. Furthermore, assays using filipin and nystatin showed down regulation of EhPCTP-L, in an apparently cholesterol-independent way. Interestingly, EhPCTP-L binds primarily to anionic phospholipids phosphatidylserine (PS) and phosphatidic acid (PA), while its mammalian counterpart HsPCTP-L binds neutral phospholipids PC and PE. The present study provides information that helps reveal the possible function and regulation of PCTP-L expression in the primitive eukaryotic parasite E. histolytica.


Subject(s)
Entamoeba histolytica/metabolism , Protozoan Proteins/isolation & purification , Protozoan Proteins/metabolism , Acetylation , Amino Acid Sequence , Animals , Caveolin 1/immunology , Cell Membrane/metabolism , Chick Embryo , Cholesterol/metabolism , Cross Reactions , Cytoplasm/metabolism , Entamoeba histolytica/drug effects , Entamoeba histolytica/genetics , Filipin/pharmacology , Molecular Sequence Data , Nystatin/pharmacology , Phosphatidylcholines/metabolism , Phospholipid Transfer Proteins/immunology , Phospholipid Transfer Proteins/isolation & purification , Phospholipid Transfer Proteins/metabolism , Phosphoproteins/chemistry , Protein Conformation , Protein Structure, Tertiary , Protozoan Proteins/genetics , Protozoan Proteins/immunology
8.
Chem Res Toxicol ; 26(6): 918-25, 2013 Jun 17.
Article in English | MEDLINE | ID: mdl-23659204

ABSTRACT

Human phospholipid scramblase 1(hPLSCR1) is a transmembrane protein involved in bidirectional scrambling of plasma membrane phospholipids during cell activation, blood coagulation, and apoptosis in response to elevated intracellular Ca(2+) levels. Pb(2+) and Hg(2+) are known to cause procoagulant activation via phosphatidylserine exposure to the external surface in erythrocytes, resulting in blood coagulation. To explore its role in lead and mercury poisoning, hPLSCR1 was overexpressed in Escherichia coli BL21 (DE3) and purified using affinity chromatography. The biochemical assay showed rapid scrambling of phospholipids in the presence of Hg(2+) and Pb(2+). The binding constant (Ka) was calculated and found to be 250 nM(-1) and 170 nM(-1) for Hg(2+) and Pb(2+), respectively. The intrinsic tryptophan fluorescence and far ultraviolet circular dichroism studies revealed that Hg(2+) and Pb(2+) bind to hPLSCR1 and induce conformational changes. hPLSCR1 treated with protein modifying reagent N-ethylmaleimide before functional reconstitution showed 40% and 24% inhibition in the presence of Hg(2+) and Pb(2+), respectively. This is the first biochemical evidence to prove the above hypothesis that hPLSCR1 is activated in heavy metal poisoning, which leads to bidirectional transbilayer movement of phospholipids.


Subject(s)
Lead/pharmacology , Lipid Bilayers/metabolism , Mercury/pharmacology , Phospholipid Transfer Proteins/metabolism , Phospholipids/metabolism , Humans , Lipid Bilayers/chemistry , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/isolation & purification , Phospholipids/chemistry
9.
Biol Chem ; 393(10): 1173-81, 2012 Oct.
Article in English | MEDLINE | ID: mdl-23089641

ABSTRACT

Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli. Functional assay showed that Ca2+, Mg2+, and Zn2+ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fluorescence spectroscopy revealed that Ca2+ and Mg2+ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca2+ has more affinity than Mg2+ and Zn2+. Stains-all studies further confirm that hPLSCR4 is a Ca2+-binding protein. Point mutation (Asp290→Ala) in hPLSCR4 decreased the Ca2+-binding affinity as well as Tb3+ luminescence, suggesting residues of the predicted Ca2+-binding motif are involved in Ca2+ binding. Functional reconstitution with (Asp290→Ala) mutant led to ~50% and ~40% decrease in scramblase activity in the presence of Ca2+ and Mg2+, respectively.


Subject(s)
Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/metabolism , Anilino Naphthalenesulfonates/chemistry , Calcium/metabolism , Calcium/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions/drug effects , Mutagenesis, Site-Directed , Mutation , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/isolation & purification , Spectrometry, Fluorescence
10.
J Ind Microbiol Biotechnol ; 39(7): 1041-8, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22389205

ABSTRACT

Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca(2+)) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca(2+) ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques.


Subject(s)
Escherichia coli/cytology , Escherichia coli/genetics , Inclusion Bodies/enzymology , Phospholipid Transfer Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Sarcosine/analogs & derivatives , Chromatography, Affinity , Chromatography, Gel , Circular Dichroism , Escherichia coli/metabolism , Fluorescence , Humans , Inclusion Bodies/chemistry , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/metabolism , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarcosine/chemistry , Tryptophan/analysis
11.
Proc Natl Acad Sci U S A ; 106(39): 16586-91, 2009 Sep 29.
Article in English | MEDLINE | ID: mdl-19805341

ABSTRACT

Type-IV P-type ATPases (P4-ATPases) are putative phospholipid translocases, or flippases, that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. In addition, the activity of Drs2p, a P4-ATPase from Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi and endosomes, suggesting a role for phospholipid translocation in vesicle budding. Drs2p is necessary for translocation of a fluorescent phosphatidylserine analogue across purified Golgi membranes. However, a flippase activity has not been reconstituted with purified Drs2p or any other P4-ATPase, so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. Here, we show that Drs2p can catalyze phospholipid translocation directly through purification and reconstitution of this P4-ATPase into proteoliposomes. The noncatalytic subunit, Cdc50p, also was reconstituted in the proteoliposome, although at a substoichiometric concentration relative to Drs2p. In proteoliposomes containing Drs2p, a phosphatidylserine analogue was actively flipped across the liposome bilayer to the outer leaflet in the presence of Mg(2+)-ATP, whereas no activity toward the phosphatidylcholine or sphingomyelin analogues was observed. This flippase activity was mediated by Drs2p, because protein-free liposomes or proteoliposomes reconstituted with a catalytically inactive form of Drs2p showed no translocation activity. These data demonstrate for the first time the reconstitution of a flippase activity with a purified P4-ATPase.


Subject(s)
Adenosine Triphosphatases/metabolism , Calcium-Transporting ATPases/metabolism , Phospholipid Transfer Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/isolation & purification , Calcium-Transporting ATPases/isolation & purification , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/isolation & purification , Phospholipids/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/isolation & purification
12.
Biochim Biophys Acta ; 1791(8): 781-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19393763

ABSTRACT

Circulatory phospholipid transfer protein (PLTP) has two major functions: 1) transfer of phospholipids towards HDL particles; and 2) modulation of HDL size and composition via the HDL conversion process. In the laying hen (Gallus gallus), the massive oocyte-targeted lipid flow is achieved through the concerted actions of lipases, lipid transfer proteins, and relatives of the LDL receptor family. The aim of the study was to gain insights into the structure and functions of chicken PLTP. The results demonstrate that PLTP is highly conserved from chicken to mammals, as (i) chicken PLTP is associated with plasma HDL; (ii) it clearly possesses phospholipid transfer activity; (iii) it is inactivated at +58 degrees C; and (iv) it mediates conversion of avian and human HDL into small prebeta-mobile HDL and large fused alpha-mobile HDL particles. Our data show that HDL from different chicken models is similar in chemical and physical properties to that of man based on PLTP activity, cholesterol efflux, and HDL conversion assays. In contrast to mammals, PLTP-facilitated HDL remodeling did not enhance cholesterol efflux efficiency of chicken HDL particles.


Subject(s)
Chickens/metabolism , Cholesterol/metabolism , Lipoproteins, HDL/metabolism , Macrophages/metabolism , Phospholipid Transfer Proteins/metabolism , Animals , Biological Transport , Cholesterol/blood , Cloning, Molecular , Gene Expression Regulation , Humans , Immunoelectrophoresis , Molecular Sequence Data , Molecular Weight , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/isolation & purification , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time Factors
13.
Biotechnol Lett ; 30(12): 2131-7, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18629440

ABSTRACT

Human phospholipid scramblase 1 (hPLSCR1) scrambles plasma membrane phospholipids during cell activation, blood coagulation and apoptosis. It was over-expressed in E. coli with a histidine tag and purified from the inclusion bodies (*30 mg/l culture broth) under denaturing conditions using 8 M urea. The denatured hPLSCR1 refolded into its native configuration when urea was removed as shown by a 10-fold increase in its intrinsic fluorescence. Active hPLSCR1 showed scrambling activity in vitro after reconstituting in proteoliposomes. hPLSCR1 showed higher rates of scrambling activity for phosphatidylethanolamine than phosphatidylcholine. Binding studies with the calcium analogue "Stains-all" dye showed a characteristic peak, termed as the J band, at 650 nm. This is the first report on high level expression of hPLSCR1 with histidine tag in E. coli.


Subject(s)
Escherichia coli/genetics , Inclusion Bodies/chemistry , Phospholipid Transfer Proteins/metabolism , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/metabolism , Calcium , Carbocyanines/metabolism , Chromatography, Affinity , Dithionite , Escherichia coli/metabolism , Gene Expression , Humans , Nitrilotriacetic Acid/analogs & derivatives , Organometallic Compounds , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/isolation & purification , Protein Folding , Proteolipids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility , Spectrometry, Fluorescence , Urea
14.
Nucleic Acids Res ; 35(12): 4076-85, 2007.
Article in English | MEDLINE | ID: mdl-17567603

ABSTRACT

DNA topoisomerase (topo) II modulates DNA topology and is essential for cell division. There are two isoforms of topo II (alpha and beta) that have limited functional redundancy, although their catalytic mechanisms appear the same. Using their COOH-terminal domains (CTDs) in yeast two-hybrid analysis, we have identified phospholipid scramblase 1 (PLSCR1) as a binding partner of both topo II alpha and beta. Although predominantly a plasma membrane protein involved in phosphatidylserine externalization, PLSCR1 can also be imported into the nucleus where it may have a tumour suppressor function. The interactions of PLSCR1 and topo II were confirmed by pull-down assays with topo II alpha and beta CTD fusion proteins and endogenous PLSCR1, and by co-immunoprecipitation of endogenous PLSCR1 and topo II alpha and beta from HeLa cell nuclear extracts. PLSCR1 also increased the decatenation activity of human topo IIalpha. A conserved basic sequence in the CTD of topo IIalpha was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIalpha amino acids 1430-1441. These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1.


Subject(s)
Antigens, Neoplasm/metabolism , Cell Nucleus/enzymology , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Phospholipid Transfer Proteins/metabolism , Antigens, Neoplasm/analysis , Antigens, Neoplasm/isolation & purification , Binding Sites , DNA Topoisomerases, Type II/analysis , DNA Topoisomerases, Type II/isolation & purification , DNA-Binding Proteins/analysis , DNA-Binding Proteins/isolation & purification , HeLa Cells , Humans , Immunoprecipitation , Microscopy, Confocal , Phospholipid Transfer Proteins/analysis , Phospholipid Transfer Proteins/isolation & purification , Recombinant Proteins/metabolism , Two-Hybrid System Techniques
15.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 62(Pt 11): 1156-60, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-17077504

ABSTRACT

Sec14p is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the budding yeast Saccharomyces cerevisiae and is the founding member of a large eukaryotic protein superfamily. This protein catalyzes the exchange of either PtdIns or PtdCho between membrane bilayers in vitro and this exchange reaction requires no external input of energy or of other protein cofactors. Despite the previous elucidation of the crystal structure of a detergent-bound form of Sec14p, the conformational changes that accompany the phospholipid-exchange reaction remain undefined. Moreover, a structural appreciation of how Sec14p or its homologs bind their various phospholipid substrates remains elusive. Here, the purification and crystallization of yeast Sfh1p, the protein most closely related to Sec14p, are reported. A combination of electrospray ionization mass-spectrometry and collision-induced decomposition mass-spectrometry methods indicate that recombinant Sfh1p loads predominantly with phosphatidylethanolamine. Unlike phospholipid-bound forms of Sec14p, this form of Sfh1p crystallizes readily in the absence of detergent. Sfh1p crystals diffract to 2.5 A and belong to the orthorhombic primitive space group P2(1)2(1)2(1), with unit-cell parameters a = 49.40, b = 71.55, c = 98.21 A, alpha = beta = gamma = 90 degrees. One Sfh1p molecule is present in the asymmetric unit (V(M) = 2.5 A(3) Da(-1); V(s) = 50%). Crystallization of a phospholipid-bound Sec14p-like protein is a critical first step in obtaining the first high-resolution picture of how proteins of the Sec14p superfamily bind their phospholipid ligands. This information will significantly extend our current understanding of how Sec14p-like proteins catalyze phospholipid exchange.


Subject(s)
Phospholipid Transfer Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Base Sequence , Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Cloning, Molecular , Crystallography, X-Ray , DNA Primers , Mass Spectrometry , Phosphatidylinositols/metabolism , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/isolation & purification , Phospholipids/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae/isolation & purification , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification
16.
Biochemistry ; 45(16): 5367-76, 2006 Apr 25.
Article in English | MEDLINE | ID: mdl-16618126

ABSTRACT

The asymmetric transbilayer distribution of phosphatidylserine (PS) in the mammalian plasma membrane and secretory vesicles is maintained, in part, by an ATP-dependent transporter. This aminophospholipid "flippase" selectively transports PS to the cytosolic leaflet of the bilayer and is sensitive to vanadate, Ca(2+), and modification by sulfhydryl reagents. Although the flippase has not been positively identified, a subfamily of P-type ATPases has been proposed to function as transporters of amphipaths, including PS and other phospholipids. A candidate PS flippase ATP8A1 (ATPase II), originally isolated from bovine secretory vesicles, is a member of this subfamily based on sequence homology to the founding member of the subfamily, the yeast protein Drs2, which has been linked to ribosomal assembly, the formation of Golgi-coated vesicles, and the maintenance of PS asymmetry. To determine if ATP8A1 has biochemical characteristics consistent with a PS flippase, a murine homologue of this enzyme was expressed in insect cells and purified. The purified Atp8a1 is inactive in detergent micelles or in micelles containing phosphatidylcholine, phosphatidic acid, or phosphatidylinositol, is minimally activated by phosphatidylglycerol or phosphatidylethanolamine (PE), and is maximally activated by PS. The selectivity for PS is dependent upon multiple elements of the lipid structure. Similar to the plasma membrane PS transporter, Atp8a1 is activated only by the naturally occurring sn-1,2-glycerol isomer of PS and not the sn-2,3-glycerol stereoisomer. Both flippase and Atp8a1 activities are insensitive to the stereochemistry of the serine headgroup. Most modifications of the PS headgroup structure decrease recognition by the plasma membrane PS flippase. Activation of Atp8a1 is also reduced by these modifications; phosphatidylserine-O-methyl ester, lysophosphatidylserine, glycerophosphoserine, and phosphoserine, which are not transported by the plasma membrane flippase, do not activate Atp8a1. Weakly translocated lipids (PE, phosphatidylhydroxypropionate, and phosphatidylhomoserine) are also weak Atp8a1 activators. However, N-methyl-phosphatidylserine, which is transported by the plasma membrane flippase at a rate equivalent to PS, is incapable of activating Atp8a1 activity. These results indicate that the ATPase activity of the secretory granule Atp8a1 is activated by phospholipids binding to a specific site whose properties (PS selectivity, dependence upon glycerol but not serine, stereochemistry, and vanadate sensitivity) are similar to, but distinct from, the properties of the substrate binding site of the plasma membrane flippase.


Subject(s)
Adenosine Triphosphatases/metabolism , Lipids/pharmacology , Phospholipid Transfer Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/isolation & purification , Animals , Cell Line , Detergents/pharmacology , Enzyme Activation/drug effects , Gene Expression , Lipids/chemistry , Mice , Microsomes/enzymology , Molecular Structure , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/isolation & purification , Solubility/drug effects , Stereoisomerism
17.
FEBS Lett ; 580(1): 82-6, 2006 Jan 09.
Article in English | MEDLINE | ID: mdl-16343487

ABSTRACT

A proteomics approach was used to search for novel phospholipid binding proteins in Saccharomyces cerevisiae. Phospholipids were immobilized on a solid support and the lipids were probed with soluble yeast protein extracts. From this, the phosphatidic acid binding proteins were eluted and identified by mass spectrometry. Thirteen proteins were identified and 11 of these were previously unknown lipid binding proteins. The protein-lipid interactions identified would not have been predicted using bioinformatics approaches as none possessed a known lipid binding motif. A subset of the identified proteins was purified to homogeneity and determined to directly bind phospholipids immobilized on a solid support or organized into liposomes. This simple approach could be systematically applied to perform an exhaustive screen for soluble lipid binding proteins in S. cerevisiae or other organisms.


Subject(s)
Phosphatidic Acids/chemistry , Phospholipid Transfer Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Amino Acid Motifs , Cell-Free System/chemistry , Cell-Free System/metabolism , Phosphatidic Acids/metabolism , Phospholipid Transfer Proteins/isolation & purification , Phospholipid Transfer Proteins/metabolism , Protein Binding , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
18.
Biochemistry ; 44(22): 8111-6, 2005 Jun 07.
Article in English | MEDLINE | ID: mdl-15924430

ABSTRACT

The human tear fluid film consists of a superficial lipid layer, an aqueous middle layer, and a hydrated mucin layer located next to the corneal epithelium. The superficial lipid layer protects the eye from drying and is composed of polar and neutral lipids provided by the meibomian glands. Excess accumulation of lipids in the tear film may lead to drying of the corneal epithelium. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. To gain insight into the formation of tear film, we investigated whether PLTP and CETP are present in human tear fluid. Tear fluid samples were collected with microcapillaries. The presence of PLTP and CETP was studied in tear fluid by Western blotting, and the PLTP concentration was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. Size-exclusion and heparin-affinity chromatography assessed the molecular form of PLTP. PLTP is present in tear fluid, whereas CETP is not. Quantitative assessment of PLTP by ELISA indicated that the PLTP concentration in tear fluid, 10.9 +/- 2.4 microg/mL, is about 2-fold higher than that in human plasma. PLTP-facilitated phospholipid transfer activity in tears, 15.1 +/- 1.8 micromol mL(-)(1) h(-)(1), was also significantly higher than that measured in plasma. Inactivation of PLTP by heat treatment (+58 degrees C, 60 min) or immunoinhibition abolished the phospholipid transfer activity in tear fluid. Size-exclusion chromatography of tear fluid indicated that PLTP eluted in a position corresponding to a size of 160-170 kDa. Tear fluid PLTP was quantitatively bound to Heparin-Sepharose and could be eluted as a single peak by 0.5 M NaCl. These data indicate that human tear fluid contains catalytically active PLTP protein, which resembles the active form of PLTP present in plasma. The results suggest that PLTP may play a role in the formation of the tear film by supporting phospholipid transfer.


Subject(s)
Membrane Proteins/isolation & purification , Phospholipid Transfer Proteins/isolation & purification , Tears/chemistry , Apolipoprotein A-I/chemistry , Apolipoproteins E/chemistry , Biological Transport, Active , Blotting, Western/methods , Carrier Proteins/chemistry , Carrier Proteins/immunology , Carrier Proteins/metabolism , Cholesterol Ester Transfer Proteins , Cholesterol Esters/metabolism , Chromatography, Affinity , Chromatography, Gel , Glycoproteins/chemistry , Glycoproteins/immunology , Glycoproteins/metabolism , Heparin/metabolism , Hot Temperature , Humans , Immune Sera/chemistry , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/blood , Membrane Proteins/metabolism , Phospholipid Transfer Proteins/antagonists & inhibitors , Phospholipid Transfer Proteins/blood , Phospholipid Transfer Proteins/metabolism , Phospholipids/metabolism , Sepharose/analogs & derivatives , Sepharose/metabolism , Tears/enzymology , Tears/metabolism
19.
Methods ; 36(2): 97-101, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15893933

ABSTRACT

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. PLTP is an 80-kDa glycoprotein that is expressed/secreted by a wide variety of tissues including lung, liver, adipose tissue, brain, and muscle. PLTP mediates a net transfer of phospholipids between vesicles and plasma HDLs. It also generates from small HDL particles large fused HDL particles with a concomitant formation of small lipid-poor apolipoprotein (apo) A-I-containing particles which are thought to act as the primary acceptors of cell-derived cholesterol from peripheral tissue macrophages. Another important function of PLTP is connected to lipolysis. Its role in the transfer of surface remnants from triglyceride-rich particles, very-low-density lipoproteins, and chylomicrons, to HDL is of importance for the maintenance of HDL levels. Recent observations from our laboratory have demonstrated that in circulation two forms of PLTP are present, one catalytically active (high-activity form, HA-PLTP) and the other a low-activity form (LA-PLTP). In view of the likely relevancy of PLTP in human health and disease, reliable and accurate methods for measuring plasma/serum PLTP activity and concentration are required. In this chapter, two radiometric PLTP activity assays are described: (i) exogenous, lipoprotein-independent phospholipid transfer assay and (ii) endogenous, lipoprotein-dependent phospholipid transfer assay. In addition, an ELISA method for quantitation of serum/plasma total PLTP mass as well as HA-PLTP and LA-PLTP mass is reported in detail.


Subject(s)
Blood Chemical Analysis , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/isolation & purification , Apolipoprotein A-I/chemistry , Cholesterol/metabolism , Chylomicrons/metabolism , Enzyme-Linked Immunosorbent Assay , Glycoproteins/chemistry , Humans , Lipoproteins/chemistry , Lipoproteins, HDL/chemistry , Liposomes/chemistry , Phosphatidylcholines/chemistry , Phospholipids/chemistry , Radiometry , Tissue Distribution , Triglycerides/chemistry
20.
Article in English | MEDLINE | ID: mdl-15722041

ABSTRACT

Expanded bed absorption chromatography (EBA) was used to improve and simplify the purification of several wheat recombinant proteins. Binding and elution conditions were set to allow the purification of the over expressed protein in a single step. In comparison with our previous multi step protocol, same purity was obtained while EBA required less time (one day instead of five) and gave a higher yield (63% instead of 10%). This new procedure was then used for the successful purification of five other wheat ns-LTP. Despite their important polymorphism (identity from 44 to 97 %-pHi from 8 to 10), the EBA protocol allowed their purification in a single step.


Subject(s)
Chromatography, Affinity/methods , Plant Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Triticum/metabolism , Adsorption , Membrane Proteins/isolation & purification , Phospholipid Transfer Proteins/isolation & purification , Pichia/metabolism
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