Takahiro Nagase - Academia.edu (original) (raw)

Papers by Takahiro Nagase

Current chemical genomics, 2012

Protocadherin-24 (PCDH24) is linked to the suppression of tumor growth and the inhibition of cell... more Protocadherin-24 (PCDH24) is linked to the suppression of tumor growth and the inhibition of cell proliferation in the colon cancer cell line HCT116. We previously observed that β-catenin is localized to the plasma membrane when PCDH24 is expressed in these cells, but the molecular mechanisms by which PCDH24 induces the membrane localization of β-catenin remain largely unknown. To clarify these mechanisms, we identified molecules that interact with ectopically expressed PCDH24 in HCT116 cells using a HaloTag® pull-down assay. We found that galectin-1 and galectin-3 physically interact with PCDH24 and are retained at the plasma membrane in association with PCDH24 expression. A luciferase-based pull-down assay using HaloTag-fused galectins revealed that an intracellular region of PCDH24 (amino acids 1186-1280) is essential for this interaction. Furthermore, the over-expression of galectin-1 or -3, or the depletion of endogenous galectins by small interfering RNA modulates β-catenin tr...

We established a protocol for the prediction of the coding sequences of unidentified human genes ... more We established a protocol for the prediction of the coding sequences of unidentified human genes based on the double selection and sequence analysis of cDNA clones with inserts carrying unreported 5'-terminal sequences and with insert sizes corresponding to nearly full-length transcripts. By applying the protocol, cDNA clones with inserts longer than 2 kb were isolated from a cDNA library of

Science (New York, N.Y.), Jan 7, 1998

The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-ce... more The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target of Cdc42 and Rac1, was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.

Briefings in Functional Genomics and Proteomics, 2006

The Kazusa cDNA project pioneered an extensive sequencing project of human cDNAs in their entiret... more The Kazusa cDNA project pioneered an extensive sequencing project of human cDNAs in their entirety and focused sequencing efforts particularly on large cDNAs encoding large proteins. More than 2000 human genes, referred to as &amp;amp;amp;amp;amp;amp;amp;#39;KIAA&amp;amp;amp;amp;amp;amp;amp;#39; genes, were initially identified through this cDNA project. Since many KIAA genes still remain functionally uncharacterized, our current focus is to determine their biological functions in vivo. In this review, we describe the current status of the Kazusa mammalian cDNA resources and the future direction of the functional characterization of KIAA genes.

Structure, 2004

CYLD was originally identified as the human familial cylindromatosis tumor suppressor. Recently, ... more CYLD was originally identified as the human familial cylindromatosis tumor suppressor. Recently, it was reported that CYLD directly interacts with NEMO/IKKgamma and TRAF2 in the NF-kappaB signaling pathway. The two proteins bind to a region of CYLD that contains a Cys-box motif and the third cytoskeleton-associated protein-glycine conserved (CAP-Gly) domain. Here we report that the third CAP-Gly domain of CYLD specifically interacts with one of the two proline-rich sequences of NEMO/IKKgamma. The tertiary structure of the CAP-Gly domain shares the five-stranded beta sheet topology with the SH3 domain, which is well known as a proline-rich sequence-recognition domain. However, chemical shift mapping revealed that the peptide binding site of the CAP-Gly domain is formed without the long peptide binding loop characteristic of the SH3 domain. Therefore, CAP-Gly is likely to be a novel proline-rich sequence binding domain with a mechanism different from that of the SH3 domain.

Structure, 2006

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Re... more SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.

Nucleic Acids Research, 2004

Nucleic Acids Research, 1999

HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim ... more HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge

Nature Genetics, 2004

As a base for human transcriptome and functional genomics, we created the "full-length long Japan... more As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at ∼58% compared with a peak at ∼42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at ∼42%, relatively low compared with that of protein-coding cDNAs.

Journal of Molecular Biology, 2008

The SMARCAD1/KIAA1122 protein is structurally classified into the SWI2/SNF2 superfamily of DNA-de... more The SMARCAD1/KIAA1122 protein is structurally classified into the SWI2/SNF2 superfamily of DNA-dependent ATPases that are catalytic subunits of chromatin-remodeling complexes. Although the importance of other members of the SWR1-like subfamily in chromatin remodeling (EP400, INOC1, and SRCAP) has already been elucidated, the biological function of SMARCAD1/KIAA1122 in transcriptional regulation remains to be clarified. To gain insight into the role of this protein, we generated a specific antibody against SMARCAD1/KIAA1122 and used it for chromatin and protein immunoprecipitation assays. We employed high-resolution genome tiling microarrays in chromatin immunoprecipitation and found the binding sites of SMARCAD1/KIAA1122 in the vicinity of the transcriptional start site of 69 candidate target genes. In the protein immunoprecipitation assay, we found that endogenous SMARCAD1/KIAA1122 binds with TRIM28, a recently highlighted transcriptional regulator in the cancer field. From these findings, we propose a novel model for gene regulation via the SMARCAD1/KIAA1122 protein complex.

Journal of Biological Chemistry, 2003

CLOCK is a positive component of a transcription/translation-based negative feedback loop of the ... more CLOCK is a positive component of a transcription/translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice. We identified more than 100 genes that fluctuated from day to night and of which expression levels were decreased in Clock mutant mice. In Cry-deficient mice, the expression levels of most CLOCK-regulated genes were elevated to the upper range of normal oscillation. Most of the screened genes had a CLOCK/BMAL1 binding site (E box) in the 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-flanking region. We found that CLOCK was absolutely concerned with the circadian transcription of one type of liver genes (such as DBP, TEF, and Usp2) and partially with another (such as mPer1, mPer2, mDec1, Nocturnin, P450 oxidoreductase, and FKBP51) because the latter were damped but remained rhythmic in the mutant mice. Our results showed that CLOCK and CRY proteins are involved in the transcriptional regulation of many circadian output genes in the mouse liver. In addition to being a core component of the negative feedback loop that drives the circadian oscillator, CLOCK also appears to be involved in various physiological functions such as cell cycle, lipid metabolism, immune functions, and proteolysis in peripheral tissues.

Journal of Bacteriology, 2003

We discovered a novel small heat shock protein (sHsp) named AgsA (aggregation-suppressing protein... more We discovered a novel small heat shock protein (sHsp) named AgsA (aggregation-suppressing protein) in the thermally aggregated fraction from a Salmonella enterica serovar Typhimurium dnaK-null strain. The -10 and -35 regions upstream of the transcriptional start site of the agsA gene are characteristic of sigma(32)- and sigma(72)-dependent promoters. AgsA was strongly induced by high temperatures. The similarity between AgsA and the other two sHsps of Salmonella serovar Typhimurium, IbpA and IbpB, is rather low (around 30% amino acid sequence identity). Phylogenetic analysis suggested that AgsA arose from an ancient gene duplication or amplification at an early evolutionary stage of gram-negative bacteria. Here we show that overproduction of AgsA partially complements the DeltadnaK52 thermosensitive phenotype and reduces the amount of heat-aggregated proteins in both DeltadnaK52 and DeltarpoH mutants of Escherichia coli. These data suggest that AgsA is an effective chaperone capable of preventing aggregation of nonnative proteins and maintaining them in a state competent for refolding in Salmonella serovar Typhimurium at high temperatures.

Genomics, 1998

To identify large proteins with an EGF-like-motif in a systematic manner, we developed a computer... more To identify large proteins with an EGF-like-motif in a systematic manner, we developed a computer-assisted method called motif-trap screening. The method exploits 5'-end single-pass sequence data obtained from a pool of cDNAs whose sizes exceed 5 kb. Using this screening procedure, we were able to identify five known and nine new genes for proteins with multiple EGF-like-motifs from 8000 redundant human brain cDNA clones. These new genes were found to encode a novel mammalian homologue of Drosophila fat protein, two seven-transmembrane proteins containing multiple cadherin and EGF-like motifs, two mammalian homologues of Drosophila slit protein, an unidentified LDL receptor-like protein, and three totally uncharacterized proteins. The organization of the domains in the proteins, together with their expression profiles and fine chromosomal locations, has indicated their biological significance, demonstrating that motif-trap screening is a powerful tool for the discovery of new genes that have been difficult to identify by conventional methods.

Gene Expression Patterns, 2003

Dbl-family guanine nucleotide exchange factors (Dbl-GEFs) act as activators of Rho-like small G p... more Dbl-family guanine nucleotide exchange factors (Dbl-GEFs) act as activators of Rho-like small G proteins such as Rac1, Cdc42 and RhoA. Recently, some GEFs have been suggested to play important roles in the development of the nervous system. Here, we report a comprehensive expression profile analysis of 20 Dbl-GEFs that have yet to be well investigated. Northern analyses of murine mRNAs from brains of E13, E17, P7 and adult mice revealed expression of 18 out of 20 GEFs in some or all stages. In addition, we found that three human GEFs were highly expressed in the brain. Examination of the spatial expression patterns of five GEFs in embryos or neonatal brain by in situ hybridization revealed distinct patterns for each GEF. Our study reveals the dynamic and coordinated expression profiles of the Dbl-GEFs and provides a basic framework for understanding the function of GEFs in neural development.

Gene, 2005

Complementary DNA (cDNA) clones for human KIAA genes have been isolated as long cDNAs (&a... more Complementary DNA (cDNA) clones for human KIAA genes have been isolated as long cDNAs (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;4 kb) with unknown functions. To facilitate the functional analysis of these human clones, we have isolated and determined the structures of their respective mouse homologues (mKIAA genes). Furthermore, we have comprehensively raised antibodies against the translated mKIAA proteins in order to establish a platform for their functional analysis. Since the specificity of these antibodies is critical for subsequent analyses of protein function, here we introduce two assays utilizing mammalian cells to improve their evaluation. First, we have established a semi-high-throughput production of C-terminally FLAG epitope-tagged proteins for Western blotting using specially designed mammalian expression vectors. Secondly, we have utilized immunofluorescence staining of mouse cells to analyze the subcellular localization of endogenous mKIAA proteins. Importantly, these methods allow us to detect potential posttranslational modification of the mKIAA/KIAA proteins and to predict their biological function based on their subcellular localization.

FEBS Letters, 1999

ABSTRACT Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick senso... more ABSTRACT Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-α-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1–H4. The latter originate from precursor polypeptides H1P–H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P–H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-α-inhibitor family and is likely to be involved in DNA repair.

FEBS Letters, 1995

Import of preproteins into mltochondria requires transport machineries in both mitochondrial memb... more Import of preproteins into mltochondria requires transport machineries in both mitochondrial membranes that have been characterized in Saccharomyces cerevisiae and Neurospora crassa. By cDNA analysis, we identified a human protein of 16 kDa with significant overall homology to the fungal mitochondrial import receptor Mom19, including the three typical characteristics: a hydrophobic N-terminal segment, a tetratrico peptide motif in the middle and a negatively charged C-terminus. The human Mom19 homolog is expressed in all tissues analyzed. When synthesized in vitro, the human Mom19 homolog is targeted to isolated yeast mitochondria and specifically associates with the outer membrane receptor complex, suggesting that indeed a mitochondrial import receptor was identified.

FEBS Letters, 2000

The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remai... more The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remain to be identified. Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide range screen for GEF specificity. In this assay based on the two-hybrid system, a wild type GTPase binds to its effector only after activation by a specific GEF. We validated the YEA by activating GTPases by previously reported GEFs. We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F-actin stress fiber assembly in fibroblasts, characteristic of RhoA activation.