Littérature scientifique sur le sujet « Intracellular lipid-binding protein »

Effective cholesterol homoeostasis is essential in maintaining cellular function, and this is achieved by a network of lipid-responsive nuclear transcription factors, and enzymes, receptors and transporters subject to post-transcriptional and post-translational regulation, whereas loss of these elegant, tightly regulated homoeostatic responses is integral to disease pathologies. Recent data suggest that sterol-binding sensors, exchangers and transporters contribute to regulation of cellular cholesterol homoeostasis and that genetic overexpression or deletion, or mutations, in a number of these proteins are linked with diseases, including atherosclerosis, dyslipidaemia, diabetes, congenital lipoid adrenal hyperplasia, cancer, autosomal dominant hearing loss and male infertility. This review focuses on current evidence exploring the function of members of the ‘START’ (steroidogenic acute regulatory protein-related lipid transfer) and ‘ORP’ (oxysterol-binding protein-related proteins) families of sterol-binding proteins in sterol homoeostasis in eukaryotic cells, and the evidence that they represent valid therapeutic targets to alleviate human disease.

Inter- and intramembrane phospholipid transport processes are central features of membrane biogenesis and homeostasis. Relatively recent successes in the molecular genetic analysis of aminoglycerophospholipid transport processes in both yeast and mammalian cells are now providing important new information defining specific protein and lipid components that participate in these reactions. Studies focused on phosphatidylserine (PtdSer) transport to the mitochondria reveal that the process is regulated by ubiquitination. In addition, a specific mutation disrupts PtdSer transport between mitochondrial membranes. Analysis of PtdSer transport from the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 demonstrates the requirement for a phosphatidylinositol-4-kinase, a phosphatidylinositol-binding protein, and the C2 domain of the decarboxylase. Examination of NBD-phosphatidylcholine transport demonstrates the involvement of the prevacuolar compartment and a requirement for multiple genes involved in regulating vacuolar protein sorting for transport of the lipid to the vacuole. In intramembrane transport, multiple genes are now identified including those encoding multidrug resistant protein family members, DNF family members, ATP binding cassette transporters, and pleiotropic drug resistance family members. The scramblase family constitutes a collection of putative transmembrane transporters that function in an ATP-independent manner. The genetic analysis of lipid traffic is uncovering new molecules involved in all aspects of the regulation and execution of the transport steps and also providing essential tools to critically test the involvement of numerous candidate molecules.Key words: lipid transport, lipid sorting, membrane biogenesis, organelles, flippase.

Intracellular cholesterol transport is a complex process involving specific carrier proteins. Cholesterol-binding proteins, such as the lipid transfer protein steroidogenic acute regulatory-related lipid transfer domain-3 (STARD3), are implicated in cholesterol movements between organelles. Indeed, STARD3 modulates intracellular cholesterol allocation by reducing it from the plasma membrane and favoring its passage from the endoplasmic reticulum (ER) to endosomes, where the protein is localized. STARD3 interacts with ER-anchored partners, notably vesicle-associated membrane protein-associated proteins (VAP-A and VAP-B) and motile sperm domain-containing 2 (MOSPD2), to create ER–endosome membrane contacts. Mechanistic studies showed that at ER–endosome contacts, STARD3 and VAP proteins build a molecular machine able to rapidly transfer cholesterol. This review presents the current knowledge on the molecular and cellular function of STARD3 in intracellular cholesterol traffic.

The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[32P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.

Grateloupia elliptica (G. elliptica) is a red seaweed with antioxidant, antidiabetic, anticancer, anti-inflammatory, and anticoagulant activities. However, the anti-obesity activity of G. elliptica has not been fully investigated. Therefore, the effect of G. elliptica ethanol extract on the suppression of intracellular lipid accumulation in 3T3-L1 cells by Oil Red O staining (ORO) was evaluated. Among the eight red seaweeds tested, G. elliptica 60% ethanol extract (GEE) exhibited the highest inhibition of lipid accumulation. GEE was the only extract to successfully suppress lipid accumulation among ethanol extracts from eight red seaweeds. In this study, we successfully isolated chlorophyll derivative (CD) from the ethyl acetate fraction (EA) of GEE by high-performance liquid chromatography and evaluated their inhibitory effect on intracellular lipid accumulation in 3T3-L1 adipocytes. CD significantly suppressed intracellular lipid accumulation. In addition, CD suppressed adipogenic protein expression such as sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-α (C/EBP-α), and fatty acid binding protein 4 (FABP4). Taken together, our results indicate that CD from GEE inhibits lipid accumulation by suppressing adipogenesis via the downregulation of adipogenic protein expressions in the differentiated adipocytes. Therefore, chlorophyll from G. elliptica has a beneficial effect on lipid metabolism and it could be utilized as a potential therapeutic agent for preventing obesity.

Lipid droplets (LDs) are ubiquitously expressed organelles; the only intracellular organelles that contain a lipid monolayer rather than a bilayer. Proteins localize and bind to this monolayer as they do to intracellular lipid bilayers. The mechanism by which cytosolic LD binding proteins recognize, and bind, to this lipid interface remains poorly understood. Amphipathic α-helix bundles form a common motif that is shared between cytosolic LD binding proteins (e.g., perilipins 2, 3, and 5) and apolipoproteins, such as apoE and apoLp-III, found on lipoprotein particles. Here, we use pendant drop tensiometry to expand our previous work on the C-terminal α-helix bundle of perilipin 3 and the full-length protein. We measure the recruitment and insertion of perilipin 3 at mixed lipid monolayers at an aqueous-phospholipid-oil interface. We find that, compared to its C-terminus alone, the full-length perilipin 3 has a higher affinity for both a neat oil/aqueous interface and a phosphatidylcholine (PC) coated oil/aqueous interface. Both the full-length protein and the C-terminus show significantly more insertion into a fully unsaturated PC monolayer, contrary to our previous results at the air-aqueous interface. Additionally, the C-terminus shows a preference for lipid monolayers containing phosphatidylethanolamine (PE), whereas the full-length protein does not. These results strongly support a model whereby both the N-terminal 11-mer repeat region and C-terminal amphipathic α-helix bundle domains of perilipin 3 have distinct lipid binding, and potentially biological roles.

ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and are proposed to target intracellular lipid-transfer proteins of the oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phosphatidylinositol 4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.

Il principale obiettivo di questo lavoro di tesi è lo studio del ruolo giocato da un ponte disolfuro sulle proprietà di legame di una proteina citosolica, la Liver Bile Acid Binding Protein (L-BABP), nella quale è naturalmente presente. In particolare si vuole far luce sulle capacità della proteina di legare Acidi Biliari (BA) e sulle sue proprietà funzionali. Gli acidi biliari circolano tra il fegato e l’intestino attraverso un meccanismo definito “circolazione enteroepatica”, il quale è fortemente regolato dagli stessi acidi biliari. Gli acidi biliari sono infatti in grado di influenzare l’espressione di numerosi geni coinvolti nella loro sintesi e nel loro trasporto, mediante un legame con recettori di acidi biliari intracellulari primari, quali il recettore farnesoide X (FXR). La comprensione del meccanismo che regola l’interazione di trasportatori intracellulari con acidi biliari è un passaggio chiave per la costruzione di un modello rappresentativo del trasferimento di BAs dal citoplasma al nucleo e potrebbe essere utilizzato per lo studio di agenti terapeutici applicabili nel trattamento di disordini metabolici, quali l’obesità, il diabete di tipo 2, l’iperlipidemia e l’aterosclerosi. Per raggiungere una dettagliata descrizione dal punto di vista molecolare e della dinamica coinvolta nella formazione di un complesso ternario, tra L-BABP e due molecole di acidi biliari, è stata utilizzata la spettroscopia NMR (Nuclear Magnetic Resonance), parallelamente ad un’analisi cinetica e termodinamica, specificatamente implementata per questi studi. Nello specifico, mediante la Risonanza Magnetica Nucleare, sono state studiate le proprietà strutturali, di interazione e di dinamica di due forme di L-BABP di pollo, diverse tra loro per la presenza/assenza di un ponte disolfuro. Le interazioni proteina/ligando caratteristiche del complesso sono state studiate arricchendo alternativamente la proteina ed il ligando, con isotopi NMR attivi. La proteina è stata titolata aggiungendo concentrazioni sempre crescenti dell’acido glico-colico (GCA) e glico-chenodeossicolico (GCDA), arricchiti in 15N, in modo da poter seguire la variazione delle loro risonanze attraverso l’acquisizione e l’analisi di numerosi spettri NMR (HSQC, DOSY). I dati ottenuti hanno permesso di determinare la stechiometria di legame e i fenomeni di scambio, ma non sono risultati sufficienti per ricavare informazioni dettagliate sull’affinità, la cooperatività e i meccanismi di legame. Si è quindi deciso di analizzare la variazione dei segnali NMR in funzione della concentrazione di ligando per fare maggiore chiarezza sul meccanismo di interazione tra L-BABP e gli acidi biliari. A questo scopo, sono stati recentemente riportati, nuovi approcci NMR per lo studio delle interazioni proteina/ligandi che avvengono nella scala dei tempi dei micro- e millisecondi, che sfruttano l’analisi delle larghezze di riga ed esperimenti di “relaxation dispersion”. In particolare la combinazione di questi due approcci di indagine si sono rivelati utili per la comprensione della relazione esistente tra dinamica e funzione della proteina. Studi di rilassamento 15N, effettuati sulla proteina apo, hanno rivelato la presenza di moti lenti, nella scala dei tempi de micro- millisecondi. La principale domanda a cui si vuole rispondere è se tali moti sono essenziali per il legame con gli acidi biliari, se portano a conformazioni competenti all’inserimento dei ligandi e se sono influenzati dalla presenza del ponte disolfuro. L’analisi delle larghezze di riga, estratte dagli esperimenti di titolazione, effettuati sulla proteina arricchita isotopicamente in 15N, con successive aggiunte di GCDA, e gli esperimenti di “relaxation dispersion” hanno permesso di individuare un meccanismo di legame a più stadi e di ricavare alcune delle costanti cinetiche coinvolte.
The aim of this thesis is to understand the role played by a naturally occurring disulphide bridge on the bile acid (BA) binding and functional properties of cytosolic Liver Bile Acid Binding Protein (L-BABP). Bile acids circulate between liver and intestine through a mechanism defined as “enterohepatic circulation”, which is a tightly regulated process, particularly by BAs themselves. Indeed BAs are able to influence the expression of numerous genes involved in their synthesis and transport by binding to the primary intracellular nuclear bile acid receptor, farnesoid X receptor (FXR). Understanding the mechanism regulating the interactions of intracellular carriers with bile acid is a key step to provide a model for the transfer of BAs from cytoplasm to the nucleus and can be used to inspire design of therapeutic agents in the treatment of metabolic disorders, such as obesity, type 2 diabetes, hyperlipidaemia and atherosclerosis. To achieve a detailed molecular and dynamical description of the binding mechanism driving to the formation of the ternary complex of L-BABPs with two BA molecules, spectroscopic methods together with kinetic and thermodynamic analysis have been applied and implemented. In particular structural, dynamical and interaction properties of two forms of chicken L-BABP (cL-BABP), differing by the presence/absence of a naturally occurring disulphide bridge, have been investigated through nuclear magnetic resonance (NMR) approaches. The study of protein-ligand interactions by NMR was performed analysing complexes where, alternatively, either the protein or the ligand were isotopically labelled. 15N enriched glycocholic (GCA) and glycochenodeoxycholic acid (GCDA), two of the most important members of bile salts pool, were employed for protein titrations and their resonances followed through the acquisition and analysis of several NMR experiments (HSQC, DOSY). The obtained results shed light on binding stoichiometry and ligand exchange phenomena but were not sufficient to derive detailed information on affinity, cooperativity and binding mechanism. Thus NMR lineshape analysis as a function of ligand concentration was chosen as an appropriate tool to investigate the complex interaction mechanism within the cL-BABP/BA system. In this line, new NMR approaches have been recently described which allow a reliable and sensitive investigation of ligand binding events occurring on microsecond to millisecond (μs-ms) time scales using lineshape and relaxation dispersion experiments[1]. Particularly, the combination of these NMR methods can be useful in the study of complex multi-step mechanisms, allowing the correlation between protein dynamics and function[2]. 15N relaxation studies, performed on the apo-protein, revealed the presence of slow motions occurring on the microseconds-milliseconds timescale. The central question to be addressed is here whether these motions are essential for ligand uptake, how they can eventually lead to conformations competent for binding and how they are influenced by the presence of the disulfide bridge. The analysis of titration experiments of 15N labelled protein with unlabelled GCDA through lineshape analysis and relaxation dispersion allowed to define a multi-step binding mechanism for bile salt binding to liver BABPs and to provide an estimate of the kinetics involved.

Cholesterol is an essential molecule that mediates a myriad of critical cellular processes, such as signal transduction in eukaryotes, membrane fluidity, and steroidogenesis. As such it is not surprising that cholesterol homeostasis is tightly regulated, striking a precise balance between endogenous synthesis and regulated uptake/efflux to and from extracellular acceptors. In mammalian cells, sterol efflux is a key component of the homeostatic equation and is mediated by members of the ATP binding cassette (ABC) transporter superfamily. ATP-binding cassette (ABC) transporters represent a group of evolutionarily highly conserved cellular transmembrane proteins that mediate the ATP-dependent translocation of substrates across membranes. Members of this superfamily, ABCA1 and ABCG1, are key components of the reverse cholesterol transport pathway. ABCG1 acts in concert with ABCA1 to maximize the removal of excess cholesterol from cells by promoting cholesterol efflux onto mature and nascent HDL particles, respectively. To date, mammalian ABC transporters are exclusively associated with efflux of cholesterol. In Saccharomyces cerevisiae, we have demonstrated that the opposite (i.e inward) transport of sterol in yeast is also dependent on two ABC transporters (Aus1p and Pdr11p). This prompts the question what dictates directionality of sterol transport by ABC transporters. The main focus of this study is to define the parameters that result in sterol movement across membranes. The comparison between these contrasting states (outward v. inward transport of the same substrate) will allow us to dissect whether sterol transport across the plasma membrane is defined by the molecule (i.e. the ABC transporter) or by microenvironment (i.e. the status of other proteins and lipids) in which it resides. We have developed the model eukaryote Saccharomyces cerevisiae as a tool to understand the mechanisms that influence ABC-transporter mediated movement of sterols. Specifically, we expressed murine ABCG1 (mABCG1) in yeast and assessed how changes in the intracellular sterol environment affect movement of sterols by this transporter. We found that expression of mABCG1 is able to vary (both increase and decrease) the concentration of exogenous sterols in the cell in response to intracellular sterol changes. We also found that yeast members of the ABCG subfamily, Aus1p and Pdr11p are able to promote either influx of cholesterol or efflux of a cholesterol derivative depending on the sterol context of the cell. This is the first example of an ABC transporter mediating bi-directional transport. These data suggest that direction of transport is not a static property of the transporter but rather can adapt in response to changes in the intracellular microenvironment. In addition to sterols we also found that proteins in the microenvironment may also influence direction of transport. Specifically, we found that the yeast sterol esterifying enzyme Are2p, physically interacts with the ABC transporters Aus1p and Pdr11p. Furthermore, all three proteins were found to co-localize to detergent resistant membrane microdomains. Deletion of either ABC transporter resulted in Are2p re-localization from DRMs to a detergent soluble fraction as well as a significant decrease in the percent of sterol esterified. This phenomenon is evolutionarily conserved in the murine lung where ABCG1 and ACAT1 were observed to co-localize with flotillin-1, a marker of DRMs. We propose that co-localization and complex formation of sterol esterification enzymes and ABC transporters in DRMs reflects a novel mechanism that directs membrane sterols to the esterification reaction. The studies presented in this thesis provide evidence that direction of transport is not a static inherent property of the transporter, but rather that it is mutable and influenced by surrounding sterols and proteins. The data provided here offers further insight as to how ABC transporters move cholesterol from the membrane and therefore may provide a platform for innovative strategies to combat atherosclerosis.

The original endocrine physiologists viewed hormones as responses to homoeostatic challenge, any signal a call to arms; the word is thus derived from the classical Greek ωρμαειν‎—‘to arouse’. In the twenty-first century a hormone is a molecule—small or large, protein or lipid—secreted in a regulated fashion from one organ and acting on another. The definition is firmly based on the anatomy of the seventeenth century, the histology of the nineteenth, and the physiology of the twentieth. It has been shaped by convention and clinical specialization: gut hormones are the marches between endocrinology and gastroenterology, and the adrenal medulla the territory of the cardiovascular physician. It has been refined by concepts of paracrine—where the secretion of one cell type in a tissue acts on another cell type in the same tissue—and autocrine, where a particular cell type both secretes and responds to a particular signal. Inherent in the concepts of paracrine and autocrine are that the signal is not secreted into blood or lymph, to be distributed more or less throughout the body, but is made locally to act locally. A very good example of a signalling system with both paracrine and autocrine activities is the neuronal synapse. Inherent in the concept of the signal is that of a receptor: a signal without a receptor is the sound of one hand clapping. Inherent in the concept of a receptor are two functions: that of being able to discriminate between different signals, and to propagate the signal by activating cell membrane or intracellular signal transduction pathways. Discrimination by a receptor between different circulating potential signals is, in the first instance, a function of the likelihood of a particular signal being able to interact with the receptor, for a period of time sufficient to alter the confirmation of the receptor and thus to trigger propagation. This interaction is commonly referred to as binding, and thus the circulating hormone as a ligand (that which is bound). If the structures of ligand and receptors are such that the initial interaction is followed by formation of strong intermolecular bonds between the two, lessening the possibility of dissociation and the receptor returning to an unliganded state, the receptor is said to have high affinity for the ligand (and vice versa). If the binding is followed by propagation of the ‘appropriate’ signal the ligand is classified as an agonist, or active hormone; if a molecule occupies the binding site on the receptor but does not so alter its structure as to propagate a signal, it is classified as a hormone antagonist (and often, by extension, a receptor antagonist). In the past couple of decades, the concepts of ‘agonist’ and ‘antagonist’ have needed to be refined, as noted subsequently in this chapter.

The progression of acute HCV infection to chronic disease and subsequent extrahepatic comorbidities involve both viruses and host cellular proteins interactions as well as insurrection or subjection of cell signaling and metabolic pathways in infected cells. This interaction between host-specific factors and the hepatitis C genome also weakens or impairs other physiological or metabolic regulatory roles of the hepatocytes. Several host cell proteins promote hepatitis C infection through binding to HCV nonstructural proteins (e.g., PPP2R5D). Some studies also found cytokine (e.g., IL-10, IL-6, TNF-α, and TGF-β1) gene polymorphisms to be highly associated with chronic hepatitis C (CHC) infection progression, whereas, polymorphism in some host genes (e.g., PNPLA3, ADAR-1, and IFIH1) are found to be actively involved in the induction of advanced liver fibrosis in patients co-infected with HIV-1/HCV. Host lipid metabolism reprogramming through host lipid regulators (e.g., ANGPTL-3 and 4) is also considered essential for CHC progression to severe liver disease (e.g., cirrhosis and HCC). Several microRNAs (e.g., miR-122, miR135a) are supposed to be key mediators of HCV infection progression and development of HCC in infected individuals and associated hepatic comorbidities. In chapter 1, we have illustrated the potential roles of virus-specific proteins in HCV molecular pathogenesis. Herein, we will elucidate the host-specific culprits that subvert, impede or disrupt host cells' communications, cell signaling, and metabolic pathways to propagate HCV infection. We will also elaborate that how the subversion of infected host-cell signaling and metabolic pathways disrupt cellular networks to evolve advanced fibrosis and hepatocarcinogenesis in HCV-infected individuals.

Endothelium is important to maintain blood fluidity preventing coagulation. Glycosaminoglycan in the endothelial cell plasma membrane has been thought to prevent activation of blood coagulation. Heparin-like compound, which is a potent anticoagulant activity, has been localized on the surface of the cultured endothelial cells. Anticoagulant action associated with thrombomodulin, which is present in endothelial cells, is another mechanism to provide hemostatic nature of endothelial cells.We wondered whether any other intracellular protein(s) is involved in coagulation. We looked for such a protein(s) in cultured bovine aortic endothelial cells. We soon found an anticoagulant activity in the soluble fraction of endothelial cells and it was partially purified. This activity was adsorbed to DEAE-Sepharose and eluted from a gel filtration column in a molecular weight range of 30,000-40,000. However, limited amounts of the cells made it difficult to purify this activity. We then chose human placenta as a substitute source of this protein and have continued the purification of this anticoagulant activity.In this communication, we describe the isolation and characterization of a placental anticoagulant protein, called "PAP", which is silmilar or possible same as the endothelial anticoaguant protein. PAP was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and mono S (Pharmacia). Approximately 20 mg of the protein was purified from one placenta. The purified protein gave a single band by SDS polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein inhibited both kaolin- and thromboplastin-induced partial thromboplastin times of normal human plasma. It also inhibited the clotting time of platelet-rich plasma induced by factor Xa, but did not affect the thrombin activity of fibrinogen-fibrin conversion. The purified protein completely inhibited the prothrombin activation by reconstituted prothrombinase. The protein neither inhibited the amidolytic activity of factor Xa nor bound factor Xa. This protein specifically bound to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that PAP inhibits coagulation through the binding to phospholipid vesicles. The study on the amino acid sequence of PAP is in progress in our laboratory. Surprisingly, the sequence analysis of the cyanogen bromide fragments revealed that PAP is a new member of the lipocortin or calpactin family. The sequences of several cyanogen bromide fragments of PAP aligns with the sequences of lipocortin I and II with over 50% identity.Since PAP interacts directly with phospholipid rather than factor Xa, other activation steps in the coagulation cascade, in which phospholipid is involved, are pro^|bly affected by PAP. These reactions are the activation of factor X by a complex of factor IXa-factor VIIIa-phospholipid-Ca++ and the activations of factor X and factor IX by a tissue factor-factor VIIa-Ca++ complex.Reutelingsperger et. al,, have reported the isolation of a novel inhibitor from arteries of human umbilical cord. This protein inhibited the prothrombin activation by prothrombinase. The authors proposed that the inhibition mechanism of this inhibitor was a competition with factor Xa for binding to phospholipid. This protein is very similar to PAP as to the mode of inhibition. The molecular weight of this inhibitor is 32,000, which is slightly smaller than PAP. With the limited chemical characterization of this protein, presently it is difficult to identify this inhibitor with PAP.At the present time, the physiological role and origin of PAP is not known. PAP may originate from the endothelium of placenta, because we have detected a PAP-like anticoagulant activity in bovine aortic endothelial cells. This activity and PAP were quite alike in the purification up to the gel filtration step. If PAP antibody recognizes the antigen in the endothelial cells, it is interesting to see whether PAP localizes on the surface or inside the cells. Nevertheless, if PAP is present in the endothelial cells, it may play an important role to maintain the hemostatic nature of endothelium. PAP may bind phospholipid components at injured sites, before coagulation factors come in contact with lipid components and initiate thrombolytic events.

To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.