[Loyter Abraham]

LOYTER, ABRAHAM

Born 1936, Lodz; Ph.D. 1966, Hebrew Univ.; Lect. 1968; Sen. Lect. 1972; Assoc. Prof. 1974; Prof. 1978.

Tel: 972-2-658-5422. e-mail: loyter@vms.huji.ac.il

Research Interests:

Translocation of proteins and nucleic acid across the nuclear envelope of animal and plant cells.

Entry of viral genome into nuclei of infected cells the use of specific synthetic peptides to block nuclear import of proteins and nucleic acids, as a way to inhibit viral infection.

Research Projects:

  1. DNA translocation across nuclear envelopes of plant cells: effect of Agrobacterium VirE2.
  2. A novel approach to viral therapy: the use of synthetic peptides to inhibit translocation of viral genome into nuclei of infected cells.

Abstract of Current Research

General

The life cycle of eukaryotic cells is absolutely dependent on translocation of biologically functional proteins into the cell nucleus in which DNA replication and RNA biosynthesis occur. Studies in animal and yeast cells suggest that nuclear import is initiated in the cytoplasm by a specific binding of the nuclear localization signal (NLS) to a cytoplasmic receptor, importin a, in heterodimer with the importin ß subunit, directs the transported molecule to and through the nuclear pore complex (NPC). Recently, the crystal structure of an animal importin a-NLS complex was determined, revealing the structural and sequence requirements for the formation of an active import substrate. However, the mechanisms by which nuclear transport of importin a-NLS complexes is controlled during development of eukaryotic organisms are still obscure. Nucleo-cytoplasmic shuttling of proteins and protein-nucleic acid complexes is an essential step in many host-pathogen interactions.

Plant Nuclear Import: Characterization of Import Specificity

In our laboratory in Jerusalem, plant cytoplasmic extracts have been shown to support the NLS-dependent nuclear import in a heterologous experimental system of permeabilized mammalian cells. Using this system, we have that backbone demonstrated that peptides mimicking the NLS signal specifically inhibit nuclear uptake of an NLS-containing protein. The proposed study will utilize these findings and approaches to achieve the following specific goals:

1. To study the biological function of the cloned and expressed Arabidopsis importin a and ß proteins in nuclear import using a heterologous system, i.e. permeabilized animal cells.

2. To specifically inactivate individual members of the importin a family and to study its effect on the resulting plant cells in tissue cultures

The significance of the proposed research is two-fold. First, it will identify and characterize the protein machinery involved in the process of plant nuclear import. Secondly, the proposed experiments will determine the molecular basis for the potential substrate specificity of nuclear transport pathways.

A novel class of anti-pathogen therapeutics: Targeting protein-protein and protein-RNA interactions with synthetic peptides to inhibit infection by HIV HTLV, TYLCV and Agrobacterium tumefaciens.

HIV-1 (The human immunodeficiency virus) encodes six regulatory and/or accessory proteins. Out of these, Tat and Rev, are involved in key events in the HIV-1 life cycle. They are both are small karyophilic proteins which act in the nuclei of the infected cells by binding to specific viral RNA elements via a similar peptide motif . The same holds for the Rex protein of HTLV(the human T-lymphotropic virus 1).

Nuclear import (72) and RNA binding of Tat and Rev as well as that of the HTLV Rex are mediated by a single active region, theArginine Rich Motif (ARM). Thus ARM has a dual function, serving both as a non-conventional NLS and as an RNA-binding domain. This fact will be exploited in our project to enable highly selective anti-HIV inhibitors without interfering with the normal host cell normal physiology.

In addition to its ability to mediate its own nuclear import and binding to the viral RNA, the Tat ARM possesses an additional unique characteristic. Several studies have demonstrated that the Tat protein is able to cross cell plasma membrane, thus gaining access to the host cell cytoplasm and the cell nucleus.

Phage display peptide libraries are used in our laboratory to obtain linear peptides that specifically interact with the ARM sequences of Tat/Rev and with that of Rex((anti-ARM).The ability of these anti-Tat/rev/Rex linear peptides to inhibit nuclear import in an un-vitro assay system are studied. Also the ability of the “anti-peptide “ to penetrate into intact cells are studied. These linear anti-ARM peptides are converted into selective Backbone cyclic peptides (BC-P)(in collaboration with Prof. Chaim Glon from the Organic Chemistry department). The goal of this part of the proposed research is to design to develop cell permeabile anti-ARM BC-P bind with high efficiency to the HIV Tat and Rev and the HTLV ARM sequences in infected cells and thus inhibit HIV-1 replication.

The same approach is used to develop peptides that will specifically bind to the NLSs of the Agrobacterium VirE2 and to that of the TYLCV coat protein. This is in order to develop peptides that wiil inhibit infection of plants by Agrobacteriun and by TYLCV (The tomato yellow curl leaf virus )

Figure 1

Recent Publications: (since 1995)

Nussbaum, O. & Loyter A. (1995) Interaction of reconstituted Sendai viral envelopes with sperm cells: reconstituted Sendai virus envelope-induced fusion-mediated introduction of foreign material into bull sperm cells. Arch. Virol. 140, 1613-1622.

Broder, Y., Stanhill, A., Zakai N., Friedler, A., Gilon, C. & Loyter, A. (1997) Translocation of NLS-BSA conjugates into nuclei of permeabilized mammalian cells can be supported by protoplast extract: An experimental system for studying plant cytosolic factors involved in nuclear import. FEBS Lett. 412, 535-539.

Friedler, A., Zakai, N., Karni, O., Baraz, L., Kotler, M., Gilon, C. & Loyter, A. (1998) A backbone cyclic peptide which mimics the nuclear localization signal of HIV-1 MA inhibits nuclear import and HIV-1 production in non-dividing cells. Biochemistry 37, 5616-5622.

Karni, O, Friedler A, Zakai N, Gilon C. & Loyter A. (1998) A peptide derived from the N-terminal region of HIV-1 Vpr promotes nuclear import in permeabilized cells: elucidation of the NLS region of the Vpr. FEBS Lett. 429, 421-425.

Friedler, A., Zakai, N., Karni, O., Friedler, D., Gilon, C., & Loyter, A. (1999) Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein. J. Mol. Biol. 289, 431-437.

Friedler; A, Luedtke,N W; Friedler, D, Loyter A, Tor, Y and Gilon C (2000) Backbone Cyclic Proteinomimeti Discriminate Between Two Functions of One Protein Sequence: Application To The HIV-1 Tat/Rev Arginine Rich Motif. J. Biol. Chem. Submitted for publication.



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