Takehisa Dewa

Photocurrent and electronic activities of oriented-His-tagged photosynthetic light-harvesting/reaction center core complexes assembled onto a gold electrode.

A polyhistidine (His) tag was fused to the C- or N-terminus of the light-harvesting (LH1)-α chain of the photosynthetic antenna core complex (LH1-RC) from Rhodobacter sphaeroides to allow immobilization of the complex on a solid substrate with defined orientation. His-tagged LH1-RCs were adsorbed onto a gold electrode modified with Ni-NTA. The LH1-RC with the C-terminal His-tag (C-His LH1-RC) on the modified electrode produced a photovoltaic response upon illumination. Electron transfer is unidirectional within the RC and starts when the bacteriochlorophyll a dimer in the RC is activated by light absorbed by LH1. The LH1-RC with the N-terminal His-tag (N-His LH1-RC) produced very little or no photocurrent upon illumination at any wavelength. The conductivity of the His-tagged LH1-RC was measured with point-contact current imaging atomic force microscopy, indicating that 60% of the C-His LH1-RC are correctly oriented (N-His 63%). The oriented C-His LH1-RC or N-His LH1-RC showed semiconductive behavior, that is, had the opposite orientation. These results indicate that the His-tag successfully controlled the orientation of the RC on the solid substrate, and that the RC produced photocurrent depending upon the orientation on the electrode.

Dicetyl phosphate-tetraethylenepentamine-based liposomes for systemic siRNA delivery.

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery.

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.

Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.

Selective Assembly of Photosynthetic Antenna Proteins into a Domain-Structured Lipid Bilayer for the Construction of Artificial Photosynthetic Antenna Systems: Structural Analysis of the Assembly Using Surface Plasmon Resonance and Atomic Force Microscopy

Two types of photosynthetic membrane proteins, the peripheral antenna complex (LH2) and the core antenna/reaction center complex (LH1-RC), play an essential role in the primary process of purple bacterial photosynthesis, that is, capturing light energy, transferring it to the RC where it is used in subsequent charge separation. Establishment of experimental platforms is required to understand the function of the supramolecular assembly of LH2 and LH1-RC molecules into arrays. In this study, we assembled LH2 and LH1-RC arrays into domain-structured planar lipid bilayers placed on a coverglass using stepwise combinations of vesicle-to-planar membrane formation and vesicle fusion methods. First, it was shown that assembly of LH2 and LH1-RC in planar lipid bilayers, through vesicle-to-planar membrane formation, could be confirmed by absorption spectroscopy and high resolution atomic force microscopy (AFM). Second, formation of a planar membrane incorporating LH2 molecules made by the vesicle fusion method was corroborated by AFM together with quantitative analysis by surface plasmon resonance (SPR). By combining planar membrane formation and vesicle fusion, in a stepwise manner, LH2 and LH1-RC were successfully organized in the domain-structured planar lipid membrane. This methodology for construction of LH2/LH1-RC assemblies will be a useful experimental platform with which to investigate energy transfer from LH2 to LH1-RC where the relative arrangement of these two complexes can be controlled.

Construction and structural analysis of tethered lipid bilayer containing photosynthetic antenna proteins for functional analysis.

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

Liposomal polyamine-dialkyl phosphate conjugates as effective gene carriers: chemical structure, morphology, and gene transfer activity.

Synthetic cationic lipids are promising transfection agents for gene therapy. We report here that polyamine conjugates of dialkyl phosphates, combined with natural lipids and assembled in the form of liposomes (polycationic liposome: PCL), possess high transfection activity in the COS-1 cell line. Furthermore, we describe the functional morphology of the PCL/DNA complexes as revealed by atomic force microscopy (AFM). The conjugates were synthesized from dialkyl phosphates (with alkyl chain lengths of 12, 14, or 16 carbons) by reaction with the polyamine molecules, spermidine, spermine, or polyethylenimine (PEI(1800)). [Dewa, T., et al. Bioconjugate Chem. 2004, 15, 824]. The PCL composed of the spermidine and C16 conjugate combined with phospholipid and cholesterol (conjugate/phospholipid/cholesterol = 1/1/1 as a molar ratio) exhibited 3.6 times higher activity than that of a popular commercial product. Systematic tests revealed clear correlations of the transgene activity with physical properties of the polyamine, in particular, that longer alkyl chains and the lower molecular weight polyamines (spermidine, spermine) favor high efficacy at the higher nitrogen/phosphate ratio = 24 (N/P, stoichiometric ratio of nitrogen in the conjugate to phosphate in DNA). The low molecular weight polyamine-based PCLs, which formed 150-400 nm particles with plasmid DNA (lipoplexes), exhibited approximately 3-fold higher gene transfer activity than micellar aggregates (lacking phospholipid and cholesterol) of the corresponding conjugate. In contrast, the PEI-based PCL formed large aggregates (approximately 1 microm), that, like the micellar aggregate form, had low activity. Activity of the low molecular weight polyamine-based PCLs increased linearly with the N/P of the lipoplex up to N/P = 24. Formation of lipoplexes was examined by agarose gel electrophoresis, dynamic light scattering (DLS), and AFM. At the lower N/P = 5, large aggregates of complex (approximately 1 microm), in which DNA molecules were loosely packed, were observed. At higher N/P, lipoplexes were converted into smaller particles (150-400 nm) having a lamellar structure, in which DNA molecules were tightly packed. Such morphological features of the lipoplex correlate with the dependence of transfection on the N/P in that the lamellar structures gave superior transfection. AFM also indicated that the lipoplexes disassembled significantly, releasing DNA, when the lipoplexes were exposed to acidic conditions (pH 4). The significance for transfection activity of the metamorphosis of bilayer lipoplexes is discussed relative to that of the less active micellar aggregate form, which is unresponsive to pH change.

The Open Gate Structure of the Membrane-Embedded KcsA Potassium Channel Viewed From the Cytoplasmic Side

Crystallographic studies of channel proteins have provided insight into the molecular mechanisms of ion channels, even though these structures are obtained in the absence of the membrane and some structural portions have remained unsolved. Here we report the gating structure of the membrane-embedded KcsA potassium channel using atomic force microscopy (AFM). The activation gate of the KcsA channel is located on the intracellular side, and the cytoplasmic domain was truncated to clear the view of this location. Once opened, the individual subunits in the tetramer were resolved with the pore open at the center. Furthermore, AFM was able to capture the previously unsolved bulge helix at the entrance. A molecular dynamics simulation revealed that the bulge helices fluctuated dramatically at the open entryway. This dynamic behavior was observed as vigorous open-channel noise in the single-channel current recordings. The role of the bulge helices in the open gate structure is discussed.

How Deep Is the Potential Well Confining a Protein in a Specific Conformation? A Single-Molecule Study on Temperature Dependence of Conformational Change between 5 and 18 K

The fluorescence excitation spectrum of a single chromophore molecule in a photosynthetic pigment-protein complex is known to change in time at liquid helium temperature. The spectral change reflects a conformational change of the protein to which the chromophore binds. This work follows the temporal behavior of the spectrum of a single chromophore in the temperature range between 5 adn 18 K. The temperature dependence reveals two types of conformational change of the protein, i.e., thermally activated motions over a potential barrier of ca. 0.1 kJ/mol and temperature-independent motions of tunneling of a proton.

Advanced cancer therapy by integrative antitumor actions via systemic administration of miR-499

Previously, we developed tetraethylenepentamine-based polycation liposomes (TEPA-PCL) as a vector for the delivery of small RNAs. In the present research, we attempted tumor-targeted delivery of miR-499 via systemic administration and evaluated the potency of this system as a therapeutic strategy to treat cancer. Lipoplexes were formed by mixing cholesterol-grafted miR-499 (miR-499-C) with TEPA-PCL. Firstly, human umbilical endothelial cells (HUVECs) and Colon 26 NL-17 mouse carcinoma cells were transfected with these lipoplexes in vitro. The results showed that miR-499 had antiangiogenic effects on the HUVECs and suppressed the secretion of vascular endothelial growth factor (VEGF) from the Colon 26 NL-17 cells. In addition, the growth of the latter cells was inhibited by transfection with miR-499-C/TEPA-PCL. For in vivo delivery of miR-499 to tumors via systemic injection, miR-499-C/TEPA-PCL were decorated with Ala-Pro-Arg-Pro-Gly (APRPG) peptide-conjugated polyethylene glycol (PEG) to prepare APRPG-PEG-modified lipoplexes carrying miR-499 (APRPG-miR-499). APRPG-miR-499 were injected into tumor-bearing mice via a tail vein, and these lipoplexes accumulated sufficiently in both angiogenic vessels and cancer cells. In addition, the expression of miR-499-target proteins and VEGF in the tumor cells was clearly suppressed by the treatment with APRPG-miR-499. Finally, the therapeutic effect of miR-499 on tumor growth was evaluated in mice. The tumor growth was significantly inhibited by the intravenous injection of APRPG-miR-499 at such a low dose as 0.5mg/kg. These results suggest that miR-499 delivered by the present system has excellent potency to treat cancer via integrative anticancer actions.