Abraham Loyter

The glycogen-amylase complex as a means of obtaining highly purified α-amylases

Abstract α-Amylases of hog pancreas, human saliva and rat parotis are specifically precipitated from crude extracts as a glycogen-enzyme complex insoluble in 40% ethanol. The specific activity (units/mg protein) of the enzymes thus precipitated approaches or is equal to that of the best crystalleine prepations. The procedure is readily carried out on a micro-scale. A method for the removal of glycogen from the complex, with the subsequent preparation of the crystalline pancreatic and parotis enzymes, is described. In the case of pancreatic amylase the enzyme-glycogen complex contains one mole of enzyme per 550 anhydroglucose units.

Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium

Proteins are involved in various equilibria that play a major role in their activity or regulation. The design of molecules that shift such equilibria is of great therapeutic potential. This fact was demonstrated in the cases of allosteric inhibitors, which shift the equilibrium between active and inactive (R and T) states, and chemical chaperones, which shift folding equilibrium of proteins. Here, we expand these concepts and propose the shifting of oligomerization equilibrium of proteins as a general methodology for drug design. We present a strategy for inhibiting proteins by “shiftides”: ligands that specifically bind to an inactive oligomeric state of a disease-related protein and modulate its activity by shifting the oligomerization equilibrium of the protein toward it. We demonstrate the feasibility of our approach for the inhibition of the HIV-1 integrase (IN) protein by using peptides derived from its cellular-binding protein, LEDGF/p75, which specifically inhibit IN activity by a noncompetitive mechanism. The peptides inhibit the DNA-binding of IN by shifting the IN oligomerization equilibrium from the active dimer toward the inactive tetramer, which is unable to catalyze the first integration step of 3′ end processing. The LEDGF/p75-derived peptides inhibit the enzymatic activity of IN in vitro and consequently block HIV-1 replication in cells because of the lack of integration. These peptides are promising anti-HIV lead compounds that modulate oligomerization of IN via a previously uncharacterized mechanism, which bears advantages over the conventional interface dimerization inhibitors.

Direct translocation of histone molecules across cell membranes

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°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.

An efficient method for reassembly of fusogenic sendai virus envelopes after solubilization of intact virions with triton X‐100

The agglutination, hemolysis, and fusion activities of intact Sendai virus particles are attributed mainly to two viral glycoproteins that are located on the viral envelope [l] . Active reassembled viral envelopes can be obtained if intact virus particles are solubilized with the detergent NP-40 and then the detergent is removed by slow dialysis [2]. During the reconstitution process, the detergent-insoluble viral nucleocapsid can be removed and substitued by exogenous proteins which, if present in the reconstitution medium, are trapped within the reassembled viral envelopes [3]. As shown in [3] . the trapped proteins can be then ‘injected’into recipient cells during viral envelope-cell fusion. The use of the reassembled viral envelopes as a vehicle for microinjection of macromolecules into animal cells, evidently has a high potential in the field of enzyme therapy and gene engineering. It appears, however, that the practical use of the above method is as yet limited by the low efficiency of the reconstitution process. According to the available data, only about 4% of the original viral hemagglutinin activity can be recovered in the reassembled envelopes [3]. This might be due to partial denaturation of the viral glycoproteins during the long dialysis (72-100 h) needed for the removal of the detergent [2,3] . It is therefore important to find out whether other detergents, besides NP40, can be used for the solubilization of Sendai virus. Recently, the detergent Triton X-100 was used for the solubilization of human erythrocyte membranes and the resolution of their anion exchange system [4] as well as for the reconstitution of the glucose

Phosphorylation and dephosphorylation of membrane proteins as a possible mechanism for structural rearrangement of membrane components

A correlation was found between dephosphorylation of chicken erythrocyte membrane proteins, aggregation of intramembrane particles, increase in the lipid bilayer phase of the membrane and exposure of membrane phospholipids toward phospholipase A and trinitrobenzene sulfonic acid. Most of the covalently bound phosphate of the membrane proteins turns over and is associated with 5 major bands. It is suggested that phosphorylation and dephosphorylation of these proteins causes changes in their charge and conformation. Such changes might affect the interaction of these proteins with the neighbouring lipids or lipoprotein complexes and results in the aggregation of intramembrane particles and relative increase in the exposed free lipid bilayer phase of the membrane.

Agrobacterium induces expression of a host F-box protein required for tumorigenicity

Agrobacterium exports DNA into plant cells, eliciting neoplastic growths on many plant species. During this process, a Skp1-Cdc53-cullin-F-box (SCF) complex that contains the bacterial virulence F-box protein VirF facilitates genetic transformation by targeting for proteolysis proteins, the Agrobacterium protein VirE2 and the host protein VIP1, that coat the transferred DNA. However, some plant species do not require VirF for transformation. Here, we show that Agrobacterium induces expression of a plant F-box protein, which we designated VBF for VIP1-binding F-box protein, that can functionally replace VirF, regulating levels of the VirE2 and VIP1 proteins via a VBF-containing SCF complex. When expressed in Agrobacterium and exported into the plant cell, VBF functionally complements tumor formation by a strain lacking VirF. VBF expression is known to be induced by diverse pathogens, suggesting that Agrobacterium has co-opted a plant defense response and that bacterial VirF and plant VBF both contribute to targeted proteolysis that promotes plant genetic transformation.

A new method for reconstitution of highly fusogenic sendai virus envelopes

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 particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.

Association of the Agrobacterium T-DNA–protein complex with plant nucleosomes

Agrobacterium represents the only natural example of transkingdom transfer of genetic information, from bacteria to plants. Before the bacterial transferred DNA (T- DNA) can integrate into the plant genome, it should be targeted to and bind the host chromatin. However, the T-DNA association with the host chromatin has not been demonstrated. Here, we study T-DNA binding to plant nucleosomes in vitro and show that it is mediated by bacterial and host proteins associated with the T-DNA. The main factor that determines nucleosomal binding of the T-DNA is the cellular VirE2-interacting protein 1 (VIP1), which functions as a molecular link between the T-DNA-associated bacterial virulence protein VirE2 and core histones. The presence of both VIP1 and VirE2 is required for association of the T-DNA with mononucleosomes in which the DNA molecule exists as a tripartite complex DNA–VirE2–VIP1. Furthermore, this nucleosome-associated ternary complex can bind another bacterial virulence factor, VirF, which is an F-box protein known to target both VirE2 and VIP1 for proteasomal degradation and uncoat the T-DNA.

Liposomes bearing a quaternary ammonium detergent as an efficient vehicle for functional transfer of TMV-RNA into plant protoplasts

Abstract Liposomes composed of phosphatidylcholine (PC) and cholesterol, and bearing the hydroxyl form of the quaternary ammonium detergent diisobutylcresoxyethoxyethyldimethylbenzylammonium (DEBDA [OH−]),were able to mediate functional transfer of tobacco mosaic virus RNA (TWV RNA) into tobacco and petunia protoplasts. Functional transfer of TMV RNA was revealed by the appearance of specific capsid polypeptides within 48 h after transfection. Transfer of TMV RNA with the aid of liposomes bearing the quaternary ammonium detergent was achieved using two alternative methods. TMV RNA was enclosed within large unilamellar liposomes composed of phosphatidylcholine and cholesterol, and bearing positively charged detergent. Incubation of such liposomes loaded with TMV RNA with plant protolasts resulted in transfer of the enclosed RNA into the recipient protoplasts. In addition, functional transfer was also achieved by a complex formed between liposomes bearing the detergent and externally added (non-enclosed) TMV RNA. Maximum transfection was obtained with a complex composed of lipids, a quaternary ammonium detergent and TMV RNA, at a ratio of 1:0.5:0.035 (w/w). Using such a complex, under optimal conditions, about 30% of the recipient protoplasts were transfected with TMV RNA. Liposomes lacking the quaternary ammonium detergent practically failed to transfer TMV RNA into tobacco or petunia protoplasts.

Development of a Functional Backbone Cyclic Mimetic of the HIV-1 Tat Arginine-rich Motif

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated “Tat11,” displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin α pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.