Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

<div><p>Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC.</p></div

Analysis of dsRBEC activity.

<p><b>A)</b> EMSA showing reduced mobility in 2% agarose of polyIC/dsRBEC complexes. Lane 1, polyIC (0.5 μg); Lanes 2–5, polyIC (0.5 μg) pre-incubated with dsRBEC (0.5–4 μg). <b>B)</b> Displacement of ¹²⁵-I EGF by dsRBEC (■) or unlabeled hEGF (▲) in A431 cells. The graph shows means ±SD from a representative experiment, performed using duplicate samples. The Kd was calculated as the mean from three independent experiments. <b>C)</b> Western blot analysis of EGFR phosphorylation following treatment of MDA-MB-468 cells with dsRBEC at various concentrations for 15 minutes.</p

dsRBEC selectively introduces polyIC into EGFR over-expressing cells.

<p><b>A)</b> Expression of EGFR in MDA-MB-468 and MCF7 cells was evaluated by FACS (left) or by western blot (right) as described in the Materials and Methods. <b>B)</b> Confocal live imaging of Cy3-polyIC internalization in MDA-MB-468 and MCF7 cells. Cy3-polyIC was delivered by dsRBEC (Upper row), or added directly to the cell culture medium (Lower row). The figure shows Cy3-polyIC localization at time 0 (before treatment) and after 120 minutes of treatment (Scale bar 20μm). <b>C)</b> Cy3-polyIC/dsRBEC and AlexaFluor647-transferrin were added to MDA-MB-468 cells simultaneously. Endosomal localization of Cy3-polyIC/dsRBEC is indicated by its strong co-localization with the recycling endosomal marker transferrin, 60 minutes after the start of treatment. Cy3-polyIC (red), transferrin (green), merge (yellow). (Scale bar 10μm).</p

qRT-PCR primer sequences.

<p>qRT-PCR primer sequences.</p

PolyIC/dsRBEC induces the expression and secretion of pro-inflammatory cytokines in MDA-MB-468 cells.

<p><b>A)</b> qRT-PCR analysis of IFN-β, CCL5, IP10 and TNFα mRNA expression following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 2 and 4 hours. Data were normalized to GAPDH and are expressed as fold change relative to vehicle-treated samples. A representative experiment out of 3 experiments is shown. Error bars represent RQ max. <b>B)</b> Protein levels of IFN-β, CCL5, IP10 and TNFα were measured by ELISA following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 24 hours. Values are averages of triplicate biological samples from one representative experiment. (***,P<0.0001 for effect of polyIC/dsRBEC vs polyIC alone, for polyIC/dsRBEC vs dsRBEC alone and for polyIC/dsRBEC vs vehicle).</p

Purification of dsRBEC.

<p><b>A)</b> dsRBEC was purified on Ni Sepharose under native conditions. Samples of total lysate (T), soluble fraction (S), unbound fraction (UB) and eluate (EL) were electrophoresed by SDS-PAGE. dsRBEC was visualized with Coomassie dye and by western blot analysis using an anti-His antibody. <b>B)</b> Samples of EL electrophoresed on 1% agarose and stained with ethidium bromide. Treatment with 10μg/ml RNase A eliminated staining. <b>C)</b> dsRBEC was purified on Ni Sepharose under denaturing conditions <b><i>(</i></b>4M urea). Samples of total lysate (T), soluble fraction (S), unbound fraction (UB) and eluate (EL) were analyzed as in (A). <b>D)</b> Equal amounts of eluted protein, isolated under native or denaturing conditions, were electrophoresed and stained with ethidium bromide. <b>E)</b> SDS-PAGE analysis of final dsRBEC purification, (15% bis-acrylamide gel stained with Coomassie dye) T, total crude lysate before purification; Ni, eluate from 4 ml Ni Sepharose column; S-75, eluate from final purification on Superdex75 (Uncropped gel can be visualized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162321#pone.0162321.s002" target="_blank">S2 Fig</a>).</p

A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides in Geobacillus stearothermophilus

Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His(6)-XynC, bound to a 220-bp fragment corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the −53 and the +34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 μM XynC. The His(6)-tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (ΔC(p)) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system