Kenji Tatematsu

Bio-nanocapsule conjugated with liposomes for in vivo pinpoint delivery of various materials.

Bio-nanocapsules (BNCs) consisting of hepatitis B virus (HBV) surface antigen (HBsAg) are approximately 50-nm hollow particles displaying a human hepatocyte-recognizing molecule (pre-S1 peptide). They have been used as an HB vaccine for the last two decades. Original BNC can incorporate various payloads (e.g., drugs, genes) by electroporation and deliver them to human hepatocytes specifically by utilizing the HBV infection mechanism. Here, we developed a new BNC conjugated with liposomes and succeeded in incorporating large materials (100-nm fluorescence-labeled polystyrene beads and >30 kbp plasmids) into the BNC-liposome complex. The complex delivered these large materials to human hepatocytes specifically ex vivo and in vivo. The transfection efficiency of the BNC-liposome complex was significantly higher than that of the original BNC. These results indicated that BNC confers the tissue- and cell-specificity on the conventional liposomes and raises new possibilities for drug delivery systems, gene delivery systems, and bio-imaging systems in vivo.

Involvement of MAPK signaling molecules and Runx2 in the NELL1-induced osteoblastic differentiation

NELL1 is an extracellular protein inducing osteogenic differentiation and bone formation of osteoblastic cells. To elucidate the intracellular signaling cascade evoked by NELL1, we have shown that NELL1 protein transiently activates the MAPK signaling cascade, induces the phosphorylation of Runx2, and promotes the rapid intracellular accumulation of Tyr‐phosphorylated proteins. Unlike BMP2, NELL1 protein does not activate the Smad signaling cascade. These findings suggest that upon binding to a specific receptor NELL1 transduces an osteogenic signal through activation of certain Tyr‐kinases associated with the Ras‐MAPK cascade, and finally leads to the osteogenic differentiation.

An automated system for high-throughput single cell-based breeding

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.

Optimal surface chemistry for peptide immobilization in on-chip phosphorylation analysis.

We investigated the optimal surface chemistry of peptide immobilization for on-chip phosphorylation analysis. In our previous study, we used a heterobifunctional cross-linker sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxalate (SSMCC) to immobilize cysteine-terminated peptides on an amine-modified gold surface. The study revealed that the phosphorylation efficiency and rate were low (only 20% at 2 h) comparing with the reaction in solution. In this study, to improve the phosphorylation efficiency, the kinase substrates were immobilized via poly(ethylene glycol) (PEG), a flexible, hydrophilic polymer. An improvement in cSrc phosphorylation was achieved (60% at 1 h) from using a PEG-inserted peptide and SSMCC. However, no phosphorylation could be detected when the peptide was immobilized with a PEG-containing cross-linker. Fluorescence-labeled peptide studies revealed that the use of longer cross-linkers resulted in lower immobilization density. We considered that the flexible PEG linker was preferable to secure high phosphorylation efficiency for the immobilized peptide, probably due to the improvement of cSrc accessibility and peptide mobility, but the immobilization protocol is critical for keeping high density of the peptide immobilization. In addition, such an accelerating effect of PEG linker against on-chip phosphorylation of an immobilized peptide may depend on kinase structures or the position of the active center, because no improvement of on-chip peptide phosphorylation was observed in protein kinase A. However, PEG linker also did not suppress the phosphorylation in protein kinase A. Thus, we concluded that SSMCC and PEGylated peptide will be a good combination for the surface chemistry of on-chip phosphorylation in peptide array.

Yeast-Based Fluorescence Reporter Assay of G Protein-coupled Receptor Signalling for Flow Cytometric Screening: FAR1-Disruption Recovers Loss of Episomal Plasmid Caused by Signalling in Yeast

Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.

Quantitative and Dynamic Analyses of G Protein-Coupled Receptor Signaling in Yeast Using Fus1, Enhanced Green Fluorescence Protein (EGFP), and His3 Fusion Protein

The mechanism of G protein‐coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone‐responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into theFUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high‐throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods.

Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae

Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNCs surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14 months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines.

Nuclear-Cytoplasmic Shuttling of a RING-IBR Protein RBCK1 and Its Functional Interaction with Nuclear Body Proteins

The intracellular localization of a RING-IBR protein, RBCK1, possessing DNA binding and transcriptional activities, has been investigated. The endogenous RBCK1 was found in both the cytoplasm and nucleus. Particularly in the nucleus, it was localized in the granular structures, most likely nuclear bodies. In contrast, the over-expressed RBCK1 was detected exclusively in the cytoplasm. When the cells were treated with leptomycin B, the over-expressed RBCK1 accumulated in the nuclear bodies. These results suggest that RBCK1 possesses the signal sequences responsible for the nuclearcytoplasmic translocation. Mutational analysis of RBCK1 has indicated that an N-terminal region containing Leu-142 and Leu-145 and a C-terminal one containing the RING-IBR domain serve as the nuclear export and localization signals, respectively. Thus, RBCK1 is a transcription factor dynamically shuttling between cytoplasm and nucleus. Furthermore, RBCK1 was found to interact with nuclear body proteins, CREB-binding protein (CBP), and promyelocytic leukemia protein (PML). Coexpression of RBCK1 with CBP significantly enhanced the transcriptional activity of RBCK1. Although PML per se showed no effect on the transcriptional activity of RBCK1, the CBP-enhanced activity was repressed by coexpression with PML, presumably through the interaction of PML and CBP. Taken together, our data demonstrate that RBCK1 is involved in transcriptional machinery in the nuclear bodies, and its transcriptional activity is regulated by nucleocytoplasmic shuttling.

Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

Identification of Ubiquitin Ligase Activity of RBCK1 and Its Inhibition by Splice Variant RBCK2 and Protein Kinase Cβ

We previously identified a RING-IBR protein, RBCK1, as a protein kinase C (PKC) β- and ζ-interacting protein, and its splice variant, RBCK2, lacking the C-terminal half including the RING-IBR domain. RBCK1 has been shown to function as a transcriptional activator whose nuclear translocation is prevented by interaction with the cytoplasmic RBCK2. We here demonstrate that RBCK1, like many other RING proteins, also possesses a ubiquitin ligase (E3) activity and that its E3 activity is inhibited by interaction with RBCK2. Moreover, RBCK1 has been found to undergo efficient phosphorylation by PKCβ. The phosphorylated RBCK1 shows no self-ubiquitination activity in vitro. Overexpression of PKCβ leads to significant increases in the amounts of intracellular RBCK1, presumably suppressing the proteasomal degradation of RBCK1 through self-ubiquitination, whereas coexpression with PKCα, PKCϵ, and PKCζ shows no or little effect on the intracellular amount of RBCK1. Taken together, the E3 activity of RBCK1 is controlled by two distinct manners, interaction with RBCK2 and phosphorylation by PKCβ. It is possible that other RING proteins, such as Parkin, BRCA1, and RNF8, having the E3 activity, are also down-regulated by interaction with their RING-lacking splice variants and/or phosphorylation by protein kinases.