五島 直樹

Cationic peptides termed protein transduction domains (PTDs) have been shown to cross biological membranes efficiently. However, proteins transduced by PTDs become entrapped within the endosomal vesicles and are not delivered into organelles. We have developed a novel protein delivery system to enhance the proton sponge effect, which results in rupture of the endosomes, by using a mixture of Wr-T transporter peptide and a commercially available cationic lipid reagent. This peptide and cationic lipid reagent mixture efficiently delivers a variety of cargo proteins into living cells by releasing them from the endosomes.

Sic family kinases (SFKs) are the earliest known family of tyrosine kinases and are widely thought to play essential roles in cellular signal transduction. Although numerous functional analyses have been performed, no study has analyzed the specificity of all SFKs on an equal platform. To gain a better understanding of SFK phosphorylation, we designed a high-throughput in vitro kinase assay on the subproteome scale using surface plasmon resonance. We reacted each of the 11 human SFKs with 519 substrate proteins, and significant phosphorylation was detected in 33.6% (1921) of the total 5709 kinase substrate combinations. A large number of novel phosphorylations were included among them. Many substrates were shown to be phosphorylated by multiple SFKs, which might reflect functional complementarity of SFKs. Clustering analysis of phosphorylation results grouped substrates into 10 categories, while the similarity of SFK catalytic specificity exhibited no significant correlation with that of amino acid sequences. In silico predictions of SRC-specific phosphorylation sites were not consistent with experimental results, implying some unknown SRC recognition modes. In an attempt to find biologically meaningful novel substrates, phosphorylation data were integrated with annotation data. The extensive in vitro data obtained in this study would provide valuable clues for further understanding SFK-mediated signal transduction.

There are several human genes that may encode proteins whose functions remain unknown. To find clues to their functions, we used the mutant yeast defective in Mad2, a component of the spindle checkpoint complex. Phenotypes that were provoked by the expression of a human C18orf26 protein in the mutant yeast encouraged further characterization of this protein in human cells. This protein was designated dynAP (dynactin-associated protein) because of its interaction with dynactin subunits that comprised a microtubule-based motor protein complex. The dynAP is a transmembrane protein localizing to Golgi apparatus and plasma membrane in a microtubule-dependent manner. This protein was expressed in half of human cancer cell lines but barely in normal human fibroblasts tested. The SV40-transformed fibroblasts expressed dynAP. Importantly, the expression of dynAP activated Akt (also known as protein kinase B) by promoting Ser(473) phosphorylation required for the full activation, whereas knockdown of dynAP abolished this activation. The ergosterol-related compounds identified by the yeast cell-based high-throughput screen abrogated activation of Akt and induced apoptosis in a dynAP-dependent manner. We propose a possible advantage of dynAP expression in cancer cells; the survival of cancer cells that express dynAP is supported by dynAP-induced activation of Akt, sustaining high rates of proliferation. The inactivation of dynAP by the selected compounds nullifies this advantage, and thereby, the apoptotic machinery is allowed to operate. Taken together, dynAP can be a new target for cancer therapy, and the selected chemicals are useful for developing a new class of anticancer drugs. Mol Cancer Ther; 9(11); 2934-42. (C) 2010 AACR.

Nucleotide sequences proximal to the initiation codon of a gene are known to affect the expression efficiency of that gene. We screened 10-bp random sequences upstream of the initiation codon of the zeocin-resistance gene to identify sequences that could enhance its expression in Saccharomyces cerevisiae. Of the isolated sequences, 20 sequences exhibited a common feature, i.e. ATG at the position -9 through -7, which resulted in the incorporation of three amino acids at the N-terminus of the protein. The introduction of these sequences upstream of the initiation codon increased the expression levels of zeocin-resistance protein by 2.2-6.5-fold. One of these sequences increased the expression levels of three out of four human proteins, thereby suggesting that this sequence may also enhance the expression efficiency of mammalian proteins in yeast.

To develop useful early and/or differential diagnostic markers for pulmonary adenocarcinomas, we generated monoclonal antibodies using A549 cells derived from pulmonary adenocarcinomas as an immunogen. Hybridoma supernatants were immunohistochemically screened for antibody production by AMeX-fixed and paraffin-embedded A549 cell preparations. Positive clones were monocloned twice by limiting dilutions. From a group of obtained antibodies, an antibody designated as KU-Lu-3 showed cytoplasmic staining. The antigen recognized by KU-Lu-3 was detected by modified two-dimensional immunoblotting, and was determined to be the receptor of activated C kinase 1 (RACK1). To evaluate the utility of KU-Lu-3, we immunohistochemically studied 184 cases of pulmonary carcinoma and paired normal lung tissues, using formalin-fixed and paraffin-embedded tissue microarray sections. The expression was significantly high and frequent in adenocarcinomas but was barely detected in a few squamous cell carcinomas and large cell carcinomas (p < 0.0001). Moreover, RACK1 expression was also significantly associated with the pathological stage, tumor size and lymph node status of adenocarcinoma patients, but not with tumor differentiation, or patient age and gender. These results suggest that RACK1 may be a novel differential diagnostic marker for pulmonary adenocarcinomas. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1 We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schaosaccharomyces pombe Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1 Indeed. flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics (C) 2010 Elsevier Inc. All rights reserved

To evaluate yeast as a high-throughput cell-based system for screening chemicals that may lead to drug development, 10,302 full-length human cDNAs (similar to 50% of the total cDNAs) were introduced into yeast. Approximately 5.6% (583 clones) of the cDNAs repressed the growth of yeast. Notably, similar to 25% of the repressive cDNAs encoded uncharacterized proteins. Small chemicals can be readily surveyed by monitoring their restorative effects on the growth of yeast. The authors focused on protein kinases because protein kinases are involved in various diseases. Among 263 protein kinase cDNAs (similar to 50% of the total) expressed in yeast, 60 cDNAs (similar to 23%), including c-Yes, a member of the Src tyrosine kinase family, inhibited the growth of yeast. Known inhibitors for protein kinases were examined for whether they reversed the c-Yes-induced inhibition of the yeast growth. Among 85 inhibitors tested, 6 compounds (PP2, PP1, SU6656, purvalanol, radicicol, and geldanamycin) reversed the inhibition, indicating a high specificity sufficient for validating this screening system. Human c-Yes was found to interact with Hsc82, one of the yeast chaperones. Radicicol and geldanamycin probably exerted their actions through interactions with Hsc82. These results indicate that when human proteins requiring molecular chaperones for their activities are subjected to the yeast screening system, 2 groups of chemicals may be found. The actions of one group are exerted through direct interactions with the human proteins, whereas those of the other group are mediated through interactions with chaperones. (Journal of Biomolecular Screening 2010:368-378)

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/beta-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC(50) value of 0.020 mu M and did not inhibit PPI between TCF7/beta-catenin and PAC1/PAC2 even at a concentration of 250 mu M. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library. (Journal of Biomolecular Screening 2009:970-979)

Completion of human genome sequencing has greatly accelerated functional genomic research. Full-length cDNA clones are essential experimental tools for functional analysis of human genes. In one of the projects of the New Energy and Industrial Technology Development Organization (NEDO) in Japan, the full-length human cDNA sequencing project (FLJ project), nucleotide sequences of approximately 30 000 human cDNA clones have been analyzed. The Gateway system is a versatile framework to construct a variety of expression clones for various experiments. We have constructed 33 275 human Gateway entry clones from full-length cDNAs, representing to our knowledge the largest collection in the world. Utilizing these clones with a highly efficient cell-free protein synthesis system based on wheat germ extract, we have systematically and comprehensively produced and analyzed human proteins in vitro. Sequence information for both amino acids and nucleotides of open reading frames of cDNAs cloned into Gateway entry clones and in vitro expression data using those clones can be retrieved from the Human Gene and Protein Database (HGPD, http://www.HGPD.jp). HGPD is a unique database that stores the information of a set of human Gateway entry clones and protein expression data and helps the user to search the Gateway entry clones.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Discoidin domain receptor ( DDR) is a cell- surface receptor tyrosine kinase activated by the binding of its discoidin ( DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 ( DDR2- DS domain), and identified the binding site to fibrillar collagen by transferred cross- saturation experiments. The DDR2- DS domain structure adopts a distorted jellyroll fold, consisting of eight beta-strands. The collagen-binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen- binding site suggests that the DDR2- DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen- binding mode of the DDR2- DS domain.

A new member of the piericidin family, JBIR-02, was isolated from mycelium of Streptomyces sp. ML55 together with two known piericidin derivatives, piericidin A(1) and IT-143-B. The structure was determined on the basis of spectroscopic data. JBIR-02 inhibited nuclear export of beta-arrestin in HeLa cells at the concentration of 20 mu M.

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1 beta, tumor necrosis factor-alpha, interferon-beta, interferon-gamma, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

We report a novel in vitro high-throughput (HTP) kinase assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as kinase activity screening, kinase substrate profiling, and kinase HTP screening of kinase inhibitors. (c) 2006 Elsevier Inc. All rights reserved.

The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.

The mutation frequencies attributable to -1 frameshift or one-base substitution in the structural genes coding for resistance to chloramphenicol (Cm) and tetracycline (Tc) were followed over several cycles of DNA replication, and found to differ several-fold, depending on the orientation of the gene on the plasmid with respect to the direction of (unidirectional ColE1-type) replication. The mutation frequency was higher when the reporter gene was present in the plasmid in the same orientation as the direction of the origin, i.e., when the transcription template is the lagging daughter strand, than when the gene was inserted in the opposite orientation. This significant difference in reversion frequencies of genes with different polarities was demonstrated only for a brief period of cell growth (several cycles of replication) after induction of the dnaQ49 mutator, but was not observed when an increased number of replication cycles, was permitted, most probably due to fixation of the mutation into both strands. The mutated intermediate DNA which possesses a misaligned basepair in the Cm gene was demonstrated to be replicated into two progeny DNA molecules one is the chloramphenicol-resistant (Cm(R)) DNA synthesized from the template strand having the mutation and the other is the Cm(S) DNA from the template strand without mutation. Our results suggest that replication-dependent mutagenesis may occur preferentially in the lagging strand.

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12-13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying both gyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA. © 1995 Springer-Verlag.

In the hin-mediated DNA inversion system, HU facilitates formation of the synaptic complex composed of two recombination sites spaced 996 bp apart and of the enhancer situated between them, by looping the DNA as to promote interaction of Hin invertase with the Fis enhancer factor [Johnson et al., Nature 329 (1987) 462-465]. The HU requirement for the in vivo hin-mediated inversion was demonstrated previously [Wada et al. Gene 76 (1989) 345-352 Hillyard et al. J. Bacteriol. 172 (1990) 5402-5407 Haykinson and Johnson, EMBO J. 12 (1993) 2503-2512] and in the current experiments. This HU action, however, required IHF when H-NS was present in the cell i.e., the inversion reaction of the hin-invertible DNA fragment carried by the ptCK1202R plasmid proceeded efficiently in host cells either deficient in H-NS or in the presence of both H-NS and IHF, but not in the cells depleted for IHF alone. The level of hin mRNA in mutant cells lacking HU or IHF, in which hin inversion did not occur, was normal or slightly increased. When IHF was absent, the stimulating effect of HU on in vitro DNA circle formation of a 125-bp hin fragment between hixL and the enhancer where Fis binds was inhibited by H-NS. The present study provides an example of a multi-component interaction between HU, H-NS and IHF on the hin DNA region, which contains three characteristic sites, a d(A/T)4 stretch and bent DNA site, and two putative IHF-binding sites. © 1994.

The molecular mechanism of autoregulation of expression of the hupA gene in Escherichia coli was examined. The promoter of the gene contains a palindromic sequence with the potential to form a cruciform DNA structure in which the -35 sequence lies at the base of the stem and the -10 sequence forms a single-stranded loop. An artificial promoter lacking the palindrome, which was constructed by replacing a 10 nucleotide repeat for the predicted cruciform arm by a sequence in the opposite orientation, was not subject to HU-repression. DNA relaxation induced by deleting HU proteins and/or inhibiting DNA gyrase in cells results in increased expression from the hupA promoter. We propose that initiation of transcription of the hupA gene is negatively regulated by steric hindrance of the functional promoter domains for formation of the cruciform configuration, which is facilitated at least in part by negative supercoiling of the hupA promoter DNA region. The promoter region of the hupB gene also contains a palindromic sequence that can assume a cruciform configuration. Negative regulation of this gene by HU proteins may occur by a mechanism similar to that operating for the hupA gene. © 1994 BY THE JOURNAL OF BIOCHEMISTRY.

The interactions of three forms of HU dimer from Escherichia coli with DNA were compared. The complexes formed between HU and short DNA fragments (35 to 132 bp), uncurved oligodeoxyribonucleotide (oligo) with and without 5-bp deoxyriboadenosine (dA) stretches and curved oligo with 5-bp dA stretches, were analyzed by the gel retardation technique. The binding of HU homodimers to the DNA was less efficient than that of HU heterodimer, and the HU-1 homodimer had lower DNA-binding capacity than the HU-2 homodimer. The equilibrium dissociation constant (KQ) for DNA of the HU-1 homodimer was 3 times that of the HU heterodimer. The HU dimers had two- to fourfold higher affinity for DNA duplexes containing 5-bp dA stretches, particularly for curved DNA. The binding of HU heterodimer to the DNA was inhibited more by the curved DNA competitor than the uncurved DNA competitor without dA stretches. 1993 © 1993 BY THE JOURNAL OF BIOCHEMISTRY.

We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleicotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg58 → Gly and Arg61 → Gly, and the HupBF3, HupBK2 and HupBK2 and HupBA1 proteins contained Phe47 → Thr, Lys37 → Gln and Ala30 → Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42°C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short β-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein. © 1990.

The closely related Escherichia coli genes hupA and hupB each encode a bacterial histone-like protein HU. We report here that mutator phage Mucts62 was unable to replicate in a hupA hupB double mutant, although it could replicate in hupA or hupB single mutant as efficiently as in the wild-type strain. Mucts62 was able to lysogenize the double mutant at 30°C cell killing occurred when the lysogen was incubated at 42°C, but did not result in phage production. High-frequency non-replicative integration of Mu into host genomic DNA soon after infection could not be detected in the hupAB double mutant. These results provide the evidence that HU protein is essential for replicative transposition of Mu phage in E. coli, and also participates in high-frequency conservative integration. © 1989.