Elana Hariton-Gazal

Hebrew University of Jerusalem, Yerushalayim, Jerusalem District, Israel

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Publications (6)23.66 Total impact

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    ABSTRACT: Due to its essential role in the virus life cycle, the viral regulatory protein Rev constitutes an attractive target for the development of new antiviral molecules. In this work, a series of Backbone Cyclic Peptide (BCP) analogs that bear a conformationally constrained arginine rich motif (ARM) of Rev were tested for in vitro inhibition of HIV-1 replication. We observed a potent suppression of HIV-1 replication in chronically infected T lymphocytic cells treated with Rev-BCPs. We further investigated possible mechanisms of HIV-1 inhibition and showed that Rev-BCPs interfere slightly with the nuclear import process and are very efficient in blocking a mechanism that controls Pr55(gag) and gp160(env) synthesis. Interestingly, these protein precursors are known to be encoded by mRNAs that require Rev-binding for nuclear export. In situ hybridization using a Cy-3 conjugated HIV-1 gag oligonucleotide probe indicated that Rev-BCPs prevent the intracellular accumulation of unspliced viral RNA. As a model, the most promising analog, Rev-BCP 14, was studied by molecular modeling and dynamics in order to identify its binding site on the Rev Response Element (RRE). The annealing simulation suggests that upon binding on the RRE, Rev-BCP 14 widens the distorted major groove of the viral RNA. Numerous contacts between peptide and RNA were found within the complex and some were identified as key components for the interactions. Altogether, our data indicate that the use of conformationally constrained Rev-BCPs represents a promising strategy for the development of new peptide-based therapeutic agents against HIV-1.
    Journal of Biomedical Science 10/2007; 14(5):565-84. · 2.46 Impact Factor
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    ABSTRACT: This work describes the synthesis and activity of a novel backbone cyclic (BC) peptide library based on the sequence of the HIV-1 Rev arginine-rich motif (ARM). All the peptides in the library possess the same sequence but differ in their ring-moiety properties. The BC peptides were synthesized using simultaneous multiple-peptide synthesis and were fully assembled using bis(trichloromethyl)carbonate as a coupling agent. All the peptides in the library had inhibitory effects on the binding of Rev-GFP to importin beta in vitro. Studies performed with one of the BC Rev-ARM analogues, Rev-13, demonstrated that, like its parental linear peptide, it is karyophilic; i.e., it is able to mediate the nuclear import of conjugated bovine serum albumin (BSA) molecules. The cell penetrating properties of the BC peptides were assessed utilizing an ELISA-based system. This assay provides a quantitative evaluation of cell penetration. Most of the peptides from the library were able to penetrate intact Colo-205 cells to varying degrees. Furthermore, these BC peptides were able to carry BSA into intact Colo-205 cells. In addition to its cell penetrating and binding properties, the BC Rev-13 analogue inhibited Rev-induced gene expression in HeLa cells by 60-70% in the low micromolar range and exhibited no cell toxicity. The potential of BC peptides bearing ARM domains as lead compounds for the production of anti-HIV drugs is discussed.
    Biochemistry 09/2005; 44(34):11555-66. · 3.38 Impact Factor
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    ABSTRACT: We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).
    Journal of Molecular Biology 02/2005; 345(2):387-400. · 3.91 Impact Factor
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    ABSTRACT: The present work shows that histones are able to directly cross cell plasma membranes and mediate penetration of macromolecules covalently attached to them. Adding a mixture containing the five nucleosomal histones, H1, H2A, H2B, H3 and H4, as well as each of the last four individual histones to intact HeLa and Colo-205 cultured cells resulted in cell penetration and nuclear import of these externally added histones. This was observed by fluorescent and confocal microscopy using fixed and unfixed cells, showing that penetration was not due to the fixation process. Accumulation was also estimated by a quantitative assay that did not require cell fixation and allowed neutralization of surface-bound histones. Translocation into the HeLa and Colo-205 cells occurred at 4 degrees C, in ATP-depleted cells and in cells incubated with sucrose (0.5 M) - conditions that block the endocytic pathway. Furthermore, various endocytosis inhibitors such as colchicine, nocodazole, cytochalasin D, brefeldin A, chloroquine and nystatin did not have any effect on the penetration process. Thus, cellular uptake was mostly due to direct translocation of the histones through the cell plasma membrane and not to endocytosis. The histones were also able to mediate penetration of covalently attached bovine serum albumin (BSA) molecules, indicating their potential as carriers for the delivery of macromolecules into living mammalian cells.
    Journal of Cell Science 12/2003; 116(Pt 22):4577-86. · 5.88 Impact Factor
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    ABSTRACT: Dermaseptins are a family of antimicrobial peptides that lyse target bacterial cells by destabilization of their membranes. Here we present a novel application of a peptide derived from the dermaseptin S4, S4(13). At nontoxic concentrations, fluorescently labeled S4(13) was able to penetrate intact cultured HeLa cells but essentially failed to enter their nuclei despite its low molecular weight. Covalent attachment of nuclear localization signal (NLS) motifs of the SV40-T-antigen and of the HIV-1 Rev protein (ARM) conferred karyophilic properties upon the S4(13). The resulting peptides, which were designated as PV-S4(13) and RR-S4(13) penetrated into intact HeLa cells and were able to accumulate within the cells' nuclei. In studies with digitonin-permeabilized cells, nuclear uptake of the PV-S4(13) and the RR-S4(13) peptides showed the same features that characterize active nuclear import. Nuclear import was observed at 37 degrees C, was ATP-dependent, and was inhibited by the free peptides bearing the SV40 NLS and the Rev and Tat ARMs. Microinjected S4(13) remained in the cytoplasm while microinjected RR-S4(13) was translocated into the cells' nuclei. The new type of cell-permeable "karyophilic" peptides described here may be of potential application as a lead compound for therapeutic purposes, as a tool to study nucleocytoplasmic shuttling in intact cells, and for the delivery of peptides to the nucleus.
    Biochemistry 08/2002; 41(29):9208-14. · 3.38 Impact Factor
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    ABSTRACT: In the present work we have constructed a series of backbone cyclic peptides, which differed in the amino acid residues located at the C-terminal position of the previously described BCvir peptide (A. Friedler, N. Zakai, O. Karni, Y.C. Broder, L. Baraz, M. Kotler, A. Loyter, C. Gilon, Biochemistry 37 (1998)). BCvir is a cyclic peptide, derived from the nuclear localization signal (NLS) of the human immunodeficiency virus type 1 matrix protein. The majority of the cyclic peptides described here inhibited nuclear import in vitro. The most potent inhibitors were those bearing bulky hydrophobic amino acids such as Leu, Phe or Nal (naphthyl Ala) at the C-terminus. On the other hand, peptides bearing polar amino acid residues such as Asn, Cys or a reduced amide bond were not inhibitory. The present studies demonstrate the importance of a bulky hydrophobic C-terminal side chain and an exocyclic amide bond preceding it, to the inhibitory activity of the NLS-derived BC peptides. Being only inhibitory, these BC peptides resemble classic receptor antagonists.
    Biochimica et Biophysica Acta 03/2002; 1594(2):234-42. · 4.66 Impact Factor