Abraham Loyter - Academia.edu (original) (raw)

Papers by Abraham Loyter

The FEBS journal, 2011

Several peptides that specifically bind the HIV-1 integrase (IN) and either inhibit or stimulate ... more Several peptides that specifically bind the HIV-1 integrase (IN) and either inhibit or stimulate its enzymatic activity were developed in our laboratories. Kinetic studies using 3'-end processing and strand-transfer assays were performed to study the mode of action of these peptides. The effects of the various peptides on the interaction between IN and its substrate DNA were also studied by fluorescence anisotropy. On the basis of our results, we divided these IN-interacting peptides into three groups: (a) IN-inhibitory peptides, whose binding to IN decrease its affinity for the substrate DNA - these peptides increased the K(m) of the IN-DNA interaction, and were thus inhibitory; (b) peptides that slightly increased the K(m) of the IN-DNA interaction, but in addition modified the V(max) and K(cat) values of the IN, and thus stimulated or inhibited IN activity, respectively; and (c) peptides that bound IN but had no effect on its enzymatic activity. We elucidated the approximate ...

Protein engineering, design & selection : PEDS, 2014

Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envel... more Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable His-tagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a m...

FEBS Letters, 1984

Chlorophyll a and chlorophyll b have been inserted into reconstituted envelopes of Sendai virus p... more Chlorophyll a and chlorophyll b have been inserted into reconstituted envelopes of Sendai virus particles. Fluorescence measurements indicated a high effkiency of energy transfer between the two chlorophyll molecules due to their close proximity in the viral envelope. Fusion of reconstituted, pigmented virus envelopes with various biological cell membranes at 37°C resulted in a significant decrease in the yield of energy transfer. Reduction in the efficiency of energy transfer was temperature and time dependent, and was also dependent upon the ratio between the reconstituted Sendai virus envelopes (donor) and recipient cells (acceptor). No reduction in the efficiency of energy transfer was observed when non-fusogenic, reconstituted viral envelopes were incubated with cell membranes.

Advances in biochemical psychopharmacology, 1980

Peptide …, 2008

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome.... more The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by ''shiftides'': peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Revderived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.

The HIV-1 Rev is a 116 residues protein, which has been assumed to be expressed after the integra... more The HIV-1 Rev is a 116 residues protein, which has been assumed to be expressed after the integration process is complete. 22 It bears a well-defined nuclear localization signal (NLS) and nuclear export signal (NES) sequences that enable

Biochimica Et Biophysica Acta-biomembranes, 1977

Ca 2÷ was introduced into fresh and ATP-depleted chicken erythrocytes through the aid of the iono... more Ca 2÷ was introduced into fresh and ATP-depleted chicken erythrocytes through the aid of the ionophore A-23187.

Biochimica Et Biophysica Acta-biomembranes, 1984

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to ... more A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus partides. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.

Febs Letters, 1988

Human immunodeficiency virus (HIV) was purified by sucrose gradient centrifugation and labeled wi... more Human immunodeficiency virus (HIV) was purified by sucrose gradient centrifugation and labeled with octadecylrhodamine B-chloride (R-18) under conditions resulting in 90% quenching of the fluorescence label. Incubation of R-1 I-labeled HIV (R-IS/HIV) with CD4-positive CEM and HUT-102 cells, but not with CD4-negative MLA-144 cells, resulted in fluorescence dequenching (DQ, increase in fluorescence) of 2&25%. Similar level of DQ was observed upon incubation of CEM cells with R-IS-labeled Sendai virus. DQ was observed when R-18/HIV was incubated with CD4+ cells at 37"C, but not at 4°C. Most of the increase in fluorescence occurred within 5 min of incubation at 37°C and was independent of medium pH over the range of pH 5-8. Preincubation of cells with the lysosomotropic agent NH&l had no inhibitory effect on DQ. Complete inhibition was observed when target cells were fixed with glutaraldehyde prior to R-18/HIV addition. Our results demonstrate application of membrane fluorescence dequenching method to a quantitative measurement of fusion between HIV and target cell membranes. As determined by DQ, HIV penetrates into target cells by a rapid, pH-independent, receptor-mediated and specific process of fusion between viral envelope and cell plasma membrane, quite similar to that observed with Sendai virus.

Febs Letters, 1978

[1]R. Rott and HD Klenk In: G. Poste and GL Nicolson, Editors, Cell Surface Reviews 2, North-Holl... more [1]R. Rott and HD Klenk In: G. Poste and GL Nicolson, Editors, Cell Surface Reviews 2, North-Holland, Amsterdam, New York (1977), pp. 47–81. ... [3]T. Ushida, M. Yamaizumi and Y. Okada, Nature 266 (1977), pp. 839–840. ... [4]ZI Cabantchik, JM Wolosin, H. Ginsburg ...

Febs Letters, 1998

Viral protein r (Vpr), a HIV-1 auxiliary protein which mediates nuclear import of the viral prein... more Viral protein r (Vpr), a HIV-1 auxiliary protein which mediates nuclear import of the viral preintegration complex (PIC), contains two regions, N- and C-terminal, which have been proposed to function as a nuclear localization signal (NLS). We have synthesized peptides corresponding to both regions (designated as VprN and VprC), conjugated them to bovine serum albumin (BSA), and tested their ability to mediate nuclear import in permeabilized cells. Only VprN, and not VprC, functioned as an active NLS and promoted translocation of the conjugate into nuclei. Nuclear import of the conjugate was found to be energy and temperature dependent and was inhibited by wheat germ agglutinin (WGA). However, it did not require the addition of cytosolic factors and was not inhibited by the prototypic SV40 large T-antigen NLS peptide. Our results show that Vpr harbours a non-conventional negatively charged NLS and therefore suggest that Vpr may use a distinct nuclear import pathway.

Proceedings of The National Academy of Sciences, 1977

Nascent calcium phosphate promotes the agglutination and fusion of human erythrocyte ghosts. Memb... more Nascent calcium phosphate promotes the agglutination and fusion of human erythrocyte ghosts. Membrane phospholipids of erythrocyte ghosts treated with Ca2+ and phosphate ions become exposed to attack by phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) (Bacillus cereus). Freeze-fracture pictures of fused erythrocyte ghosts show the presence of regions deficient in intramembrane particles in the protoplasmic face which we believe to be regions of fusion. Discontinuous regions of the protoplasmic and exoplasmic faces are observed, which are apparently intermediate stages in the process of fusion. Thin-section electron micrographs reveal deposits of calcium phosphate in areas of contact and fusion of ghosts. Ca2+ in the presence of N[tris(hydroxy-

Nucleic Acids Research, 1989

Febs Letters, 1998

Proceedings of The National Academy of Sciences, 1985

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycopr... more Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA.

Proceedings of The National Academy of Sciences, 1977

Plant Molecular Biology, 1988

Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in... more Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)(+) RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.

Nucleic Acids Research, 1989

Mature Xenopus oocytes were challenged with DNA constructs including plant regulatory elements, n... more Mature Xenopus oocytes were challenged with DNA constructs including plant regulatory elements, namely, the Cauliflower mosaic virus (CaMV) 35S promoter as well as the nopaline synthase (NOS) promoter and polyadenylation signal. The bacterial chloramphenicol acetyl transferase (CAT) was used as a reporter gene. When microinjected into these cells, the plant-derived DNA constructs effectively promoted CAT synthesis in a manner dependent on the presence of the plant promoters and probably also on the polyadenylation signals. Structural studies revealed that the supercoiled structures of the above DNA plasmids were much more active in supporting CAT synthesis in microinjected oocytes than their linear forms, with clear correlation between efficient gene expression and DNA topology.

The FEBS journal, 2011

Protein engineering, design & selection : PEDS, 2014

FEBS Letters, 1984

Advances in biochemical psychopharmacology, 1980

Peptide …, 2008

Biochimica Et Biophysica Acta-biomembranes, 1977

Biochimica Et Biophysica Acta-biomembranes, 1984

Febs Letters, 1988

Febs Letters, 1978

Febs Letters, 1998

Proceedings of The National Academy of Sciences, 1977

Nucleic Acids Research, 1989

Febs Letters, 1998

Proceedings of The National Academy of Sciences, 1985

Proceedings of The National Academy of Sciences, 1977

Plant Molecular Biology, 1988

Nucleic Acids Research, 1989