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 cellularbinding 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.allostery ͉ protein equilibrium ͉ shiftides ͉ peptides ͉ drug design
In plant-pathogen interactions, the host defends against the invading pathogen and the pathogen aims to suppress or subvert this defense. Whereas the defense suppression strategy is relatively well understood for many pathogens, the mechanisms by which pathogens can actively utilize the defense machinery of the host remain obscure. We report that Agrobacterium, a microorganism that elicits neoplastic growths on many plant species, induces expression of a plant defense-related F-box protein, VBF, which it incorporates into its own pathway for genetic transformation. Our data suggest that VBF may function to uncoat the bacterial transferred DNA from its associated virulence VirE2 and host VIP1 proteins via the SCFVBF pathway. Suppression of VBF elevates the intracellular content of VIP1, but renders the plant largely resistant to Agrobacterium, indicating that, in the infection pathway, VBF is functionally epistatic to VIP1. When expressed in Agrobacterium and exported into the plant cell, VBF facilitates tumor formation.
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.
Coprecipitation of DNA with calcium phosphate is a commonly used method of gene transfer in mammalian cells. We have found that DNA forms a tight complex with Ca P. and that DNA in this complex is resistant to nucleases present in serum or added externally. Under optimal conditions, virtually all of the recipient mouse LtklAprt-cells take up Ca P.-DNA complexes, as determined by fluorescent dyes specific for DNA (4',6-diaminilo-2-phenylindol dihydrochloride) or for calcium salts (chlorotetracycline). However, only a small proportion of the cells have detectable Ca Pi-DNA complexes in the nucleus. Uptake of the Ca P-DNA complexes was highly dependent upon the pH at which the Ca P.-DNA complex was formed and upon the concentration of DNA in the complex.It has been known for many years that under certain conditions, eukaryotic cells can take up large amounts ofDNA and transport it into the cell nucleus (1, 2). This phenomenon has been exploited to achieve transformation of eukaryotic cells with both selectable and nonselectable genes (1, 2, ¶). Addition of polycations, such as polyornithine (3) or DEAE-dextran (3, 4), to the DNA solution or precipitation of the added DNA on the cell surface by calcium phosphate (5) have proven useful in increasing both the uptake ofthe DNA and the frequency ofcell transformation. Despite the large amount of high molecular weight DNA taken into cells in the presence of certain facilitators (3, 6), at-best only about one in 104 cells eventually becomes transformed (7,8). This has limited transfer to those genes for which there exists a good selective system and to certain cell lines, such as mouse L cells, that function as efficient recipients (8).The detailed mechanisms by which external DNA is integrated into a foreign chromosome and expressed in a foreign environment are as yet poorly understood. However, a large body of recent work (9, 10) has begun to elucidate the process. On the other hand, essentially nothing-is known about the way by which DNA molecules cross the cellular membrane and are transported to the nucleus. The studies described here were designed to study the quantitative aspects and the mechanisms of entry of DNA into cells after coprecipitation with Ca Pi. We have used the specific fluorescent dye 4',6-diaminilo-2-phenylindol dihydrochloride, (DAPI), which specifically stains double-stranded DNA (11), and chlorotetracycline, which stains complex salts ofCa (12). We show that virtually all recipient cells take up Ca Pf-DNA complexes, which appear in the cytoplasm in a characteristic structure, and that only a few ofthe cells have detectable Ca P,-DNA complexes in the nucleus. Additionally, DNA uptake was highly dependent on the pH at which the Ca Pi precipitate was formed and upon the concentration of DNA in that, precipitate. MATERIALS AND METHODSCell Culture. The murine cell line Ltk-Aprt-was cultured in minimum essential medium, a modification (GIBCO), sup-plemented with 10% (vol/vol) fetal calf serum. In most cases, cells were plated in 35-mm or 60-mm...
BackgroundThe human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3′-end processing in which IN removes a pGT dinucleotide from the 3′ end of each viral long terminal repeat (LTR). Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication.Methodology/Principal FindingsUsing an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR.Conclusions/SignificanceThe results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.
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