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Hiroyoshi Ariga

Publications and source records attributed to Hiroyoshi Ariga.

At least 55 records · Page 3Linked to original sources
PubMed2003

EWS/ETS fusions activate telomerase in Ewing's tumors.

EWS/ETS is a chimeric protein identified in most Ewing's sarcomas. Although EWS/ETS has been shown to activate transcription as a transcription factor, the detailed targets of EWS/ETS in transformed cells have not been clarified. Herein, we demonstrate that telomerase is a new target of EWS/ETS fusions. Both telomerase activity and the expression level of telomerase reverse transcriptase (TERT) mRNA were up-regulated in NIH3T3 cells transformed by EWS/E1AF and EWS/FLI1 as well as in two Ewing's sarcoma cell lines. Luciferase assay using the TERT promoter revealed that EWS/E1AF and EWS/FLI1 function as positive regulators of TERT transcription in an ETS binding site-independent manner. EWS/ETS appeared to be included in the initiation complex of TERT transcription and to cooperate with CREB-binding protein (CBP)/p300. When EWS/FLI1 was knocked down in Ewing's sarcomas cells by RNA interference, the expression level of TERT mRNA and the telomerase activity were significantly decreased. These findings indicate that EWS/ETS fusion proteins activate human telomerase activity in Ewing's tumors through up-regulation of TERT gene expression, probably as a transcriptional coactivator.

Adenovirus E1A Proteins↗
PubMed2003

Crystallization and preliminary crystallographic analysis of DJ-1, a protein associated with male fertility and parkinsonism.

DJ-1 was identified as a novel oncogene product that transformed mouse NIH3T3 cells in cooperation with activated Ras. DJ-1 was also correlated with male infertility and parkinsonism. DJ-1 was crystallized using sodium citrate and HEPES at pH 7.5. The crystal belongs to space group P3(1) or P3(2), with unit-cell parameters a = 75.04, c = 74.88 A and contains two molecules in an asymmetric unit. An intensity data set was collected to 2.00 A resolution.

Buffers↗
PubMed2003

The crystal structure of DJ-1, a protein related to male fertility and Parkinson's disease.

DJ-1 is a multifunctional protein that plays essential roles in tissues with higher order biological functions such as the testis and brain. DJ-1 is related to male fertility, and its level in sperm decreases in response to exposure to sperm toxicants. DJ-1 has also been identified as a hydroperoxide-responsive protein. Recently, a mutation of DJ-1 was found to be responsible for familial Parkinson's disease. Here, we present the crystal structure of DJ-1 refined to 1.95-A resolution. DJ-1 forms a dimer in the crystal, and the monomer takes a flavodoxin-like Rossmann-fold. DJ-1 is structurally most similar to the monomer subunit of protease I, the intracellular cysteine protease from Pyrococcus horikoshii, and belongs to the Class I glutamine amidotransferase-like superfamily. However, DJ-1 contains an additional alpha-helix at the C-terminal region, which blocks the putative catalytic site of DJ-1 and appears to regulate the enzymatic activity. DJ-1 may induce conformational changes to acquire catalytic activity in response to oxidative stress.

Cloning, Molecular↗
PubMed2003

Immunocytochemical localization of DJ-1 in human male reproductive tissue.

DJ-1 was identified as an activated ras-dependent oncogene product, and was also found to be an infertility-related protein (contraception-associated protein 1; CAP 1) that was reduced in rat spermatozoa treated with ornidazole, one of the endocrine disrupting substances that causes reversible infertility in rats. CAP 1 is present in spermatozoa but is not detectable in the epididymal fluid of fertile rats and appears to be shed from sperm during treatment with ornidazole. To determine the functions of DJ-1 in the human reproductive system as a target protein of endocrine active substances, we identified the localization of DJ-1 in human testis, epididymis, ejaculated spermatozoa, and seminal plasma. DJ-1 was present in cells existing in the seminiferous tubules and Leydig cells. Some strong expressions were observed in Leydig cells and Sertoli cells, suggesting a relation with spermatogenesis via androgen receptor (AR). In ejaculated spermatozoa, DJ-1 existed on the surface of the posterior part of head and the anterior part of the midpiece. DJ-1 was also present on sperm flagella when the antibody penetrated the plasma membrane, suggesting that there are two putative roles in fertilization, one is binding to the egg, and the other is flagella movement. In contrast to previous findings, we detected DJ-1 in seminal plasma of fertile men. These results demonstrate that DJ-1 in human seminal plasma is not only from spermatozoa but also from the testis and epididymis. It is suggested that DJ-1 may play an important and as yet uncharacterized role in spermatogenesis and fertilization in humans.

Adult↗
PubMed2003

Molecular cloning of the mouse AMY-1 gene and identification of the synergistic activation of the AMY-1 promoter by GATA-1 and Sp1.

We have reported that a novel c-Myc binding protein, AMY-1, stimulated the transcription activity of c-Myc and was translocated from the cytoplasm to the nucleus in a c-Myc-dependent manner. AMY-1 works as an inducer of human K562 cell differentiation upon induction of AraC. To characterize the expression or functional importance of AMY-1, the genomic DNA of mouse AMY-1 was cloned and characterized. Both mouse and human genomic DNAs, the latter of which was retrieved from a human DNA database, comprise five exons spanning about 11 kb. To characterize the promoter of the mouse AMY-1 gene, a series of deletion constructs of the region upstream of the first ATG was linked to the luciferase gene, and their luciferase activities were measured in human HeLa and K562 cells. The results showed that Sp1 was essential for AMY-1 expression in both cell lines and that GATA-1 is also necessary in K562 cells. Sp1 in both cell lines and GATA-1 only in K562 cells were identified as proteins binding to these sites by a mobility shift assay. Furthermore, it was found that GATA-1 stimulated AMY-1 expression synergistically with Sp1 in ectopically expressed insect cells and that both proteins were associated in K562 cells.

Amino Acid Sequence↗
PubMed2003

The actin-binding domain of Slac2-a/melanophilin is required for melanosome distribution in melanocytes.

Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (DeltaABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

Actins↗
PubMed2003

Trypsinogen hL expressed in the human lung is a new member of the trypsinogen family.

Molecular cloning of cDNA encoding a new member of the trypsinogen family, named trypsinogen hL, was carried out by PCR using human lung cDNAs as templates. The primary structure of trypsinogen hL was found to be a prepro-protein and a catalytic triad, 64His, 108Asp and 201Ser. It was also found that trypsinogen hL is specifically expressed in the human lung, the expression level being 30-times higher than those in other tissues tested. A phylogenic tree analysis showed that trypsinogen hL is a new member of the trypsinogen family, a family of serine protease family proteins.

Base Sequence↗
PubMed2003

DJBP: a novel DJ-1-binding protein, negatively regulates the androgen receptor by recruiting histone deacetylase complex, and DJ-1 antagonizes this inhibition by abrogation of this complex.

DJ-1 was identified by us as a novel oncogene that transforms mouse NIH3T3 cells in cooperation with ras. We later identified PIAS (protein inhibitor of activated STAT)xalpha as a DJ-1-binding protein, and found that DJ-1 restored androgen receptor (AR) transcription activity that was repressed by PIASxalpha. To further characterize the function of DJ-1, we cloned cDNA encoding a novel DJ-1-binding protein, DJBP, by a yeast two-hybrid system. DJBP mRNA was found to be specifically expressed in the testis. In addition to the binding of DJBP to the COOH-terminal region of DJ-1, DJBP was also found to bind in vitro and in vivo to the DNA-binding domain of the AR in a testosterone-dependent manner and to be colocalized with DJ-1 or AR in the nucleus. Furthermore, a co-immunoprecipitation assay showed that the formation of a ternary complex between DJ-1, DJBP, and AR occurred in cells in which DJ-1 bound to the AR via DJBP. It was found that DJBP repressed a testosterone-dependent AR transactivation activity in monkey Cos1 cells by recruiting histone deacetylase (HDAC) complex, including HDAC1 and mSin3, and that DJ-1 partially restored its repressed activity by abrogating DJBP-HDAC complex. These results suggest that AR is positively regulated by DJ-1, which antagonizes the function of negative regulators, including DJBP.

Amino Acid Sequence↗
PubMed2002

Functional domains involved in the interaction between Orc1 and transcriptional repressor AlF-C that bind to an origin/promoter of the rat aldolase B gene.

The promoter of the rat aldolase B (AldB) gene functions in vivo as an origin of DNA replication in the cells in which transcription of the gene is repressed. Previously, we identified two closely related DNA-binding proteins, AlF-C1 and AlF-C2, which repressed the AldB gene promoter. We also reported that the binding site of these proteins, site C, is one of the required DNA elements of the AldB gene origin/promoter for autonomously replicating activity in transfected cells. In the present study, we show that AlF-C1 and AlF-C2 bind directly to Orc1, a subunit of the origin recognition complex (ORC). Deletion analyses revealed a functional domain in AlF-C2 for binding to Orc1, which is located separately from the DNA-binding domain. In addition, we found a novel protein-interacting domain in Orc1 required for the binding of AlF-C2, which was conserved in human, mouse and Chinese hamster, but not in Drosophila, frog and yeast. Thus, it is assumed that in mammalian cells, sequence- specific DNA-binding proteins are involved in recruiting ORC to regulate replication initiation and/or transcription repression.

Amino Acid Sequence↗
PubMed2002

AMY-1 interacts with S-AKAP84 and AKAP95 in the cytoplasm and the nucleus, respectively, and inhibits cAMP-dependent protein kinase activity by preventing binding of its catalytic subunit to A-kinase-anchoring protein (AKAP) complex.

We have reported that a novel c-Myc-binding protein, AMY-1, binds to cAMP-dependent protein kinase-anchoring protein 149 (AKAP149) and its splicing variant, AKAP84 and is localized in the mitochondria in a complex with RII, a regulatory subunit of cAMP-dependent protein kinase (PKA) (Furusawa, M., Ohnishi, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2001) J. Biol. Chem. 276, 36647-36651). In this study, we further found that AMY-1 competitively bound to either AKAP95 or AKAP84 in the nucleus and the cytoplasm, respectively, in a concentration-dependent manner of either AKAP. Like AKAP84, AMY-1 was found to bind to the RII-binding region of AKAP95 in vivo and in vitro and to make a ternary complex with RII. It was also found that the formation of the complex of AMY-1 with AKAP84/95 and RII prevented a catalytic subunit from binding to this AKAP complex, leading to suppression of PKA activity. These findings suggest that AMY-1 is an important modulator of PKA.

A Kinase Anchor Proteins↗
PubMed2002

AAT-1, a novel testis-specific AMY-1-binding protein, forms a quaternary complex with AMY-1, A-kinase anchor protein 84, and a regulatory subunit of cAMP-dependent protein kinase and is phosphorylated by its kinase.

AMY-1 has been identified by us as a c-Myc-binding protein and was found to stimulate c-Myc transcription activity. AMY-1 was also found to be associated with protein kinase A anchor protein 84/149 (S-AKAP84/AKAP149) in the mitochondria in somatic cells and sperm, suggesting that it plays a role in spermatogenesis. To determine the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human testis cDNA library, and a clone encoding a novel protein, AAT-1, was obtained. Three isoforms of AAT-1, AAT-1alpha, -beta, and -gamma, were found to be derived from an alternative splicing of the transcripts of the aat-1 gene, which was mapped at human chromosome 3q13-3q21. AAT-1 was found to be specifically expressed in the testis during the course of spermatogenesis and also to be present in the spermatid and mature sperm, as was AMY-1. AAT-1alpha was found to bind to and be colocalized in mitochondria with AMY-1 in human HeLa and mouse GC-1 cells. Furthermore, AAT-1alpha was found to bind to the N-terminal half of S-AKAP84/AKAP149 in a quaternary complex with AMY-1 and a regulatory subunit (RII) of cAMP-dependent kinase (PKA), in which AAT-1alpha was associated with RII via S-AKAP84/AKAP149, in rat testis and HeLa cells. It was then found that AAT-1alpha weakly stimulated a phosphorylation activity of PKA and also that AAT-1 itself was phosphorylated by PKA in vivo and in vitro. These results suggest that both AAT-1 and AMY-1 play roles in spermatogenesis.

A Kinase Anchor Proteins↗
PubMed2002

AMAP-1, a novel testis-specific AMY-1-binding protein, is differentially expressed during the course of spermatogenesis.

AMY-1 has been identified by us as a c-Myc-binding protein and was found to stimulate c-Myc transcription activity. AMY-1 was also found to be associated with AKAP84/149 in the mitochondria in somatic cells and sperm, suggesting that it plays a role in spermatogenesis. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human testis cDNA library, and a clone encoding a novel protein, AMAP-1, was obtained. The amap-1 gene was mapped at human chromosome 17q21. AMY-1 was found to bind to and be colocalized with AMAP-1 in human 293T and HeLa cells. AMAP-1 was found to be specifically expressed in the testis and expressed post-meiotically in the testis, as was AMY-1. These results suggest that both AMAP-1 and AMY-1 play roles in spermatogenesis.

Amino Acid Sequence↗
PubMed2002

Distribution of extracellular matrix tenascin-X in sciatic nerves.

Tenascin-X (TNX) is an extracellular matrix protein that is highly expressed in the peripheral nervous system as well as muscular tissues, especially the heart and skeletal muscle. However, the expression manner and the physiological role of TNX in the peripheral nervous system have not been fully investigated. In this study, we elucidated its distribution in adult mouse sciatic nerves by immunohistochemical staining. TNX was found to be localized in the perineurium and the endoneurium of sciatic nerve fibers. To examine the physiological role of TNX, we investigated sciatic nerves of TNX-deficient mice that are viable and fertile and have no obvious deficits in general performance. The thickness of myelin sheaths and the size of the individual axons in these mice appeared normal. The ultrastructure of the sciatic nerves of TNX-deficient mice were similar to those of wild-type mice. Thus, the lack of a discernible phenotype in the sciatic nerves of TNX-deficient mice suggests that TNX has either a redundant or a very subtle function in the macromolecular organization in the peripheral nerve.

Animals↗
PubMed2002

Synaptotagmin-like protein 5: a novel Rab27A effector with C-terminal tandem C2 domains.

Synaptotagmin-like proteins 1-4 (Slp1-4) are new members of the carboxyl-terminal-type (C-type) tandem C2 proteins and are classified as a subfamily distinct from the synaptotagmin and the Doc2 families, because the Slp family contains a unique homology domain at the amino terminus, referred to as the Slp homology domain (SHD). We previously showed that the SHD functions as a binding site for Rab27A, which is associated with human hemophagocytic syndrome (Griscelli syndrome) [J. Biol. Chem. 277 (2002) 9212; J. Biol. Chem. 277 (2002) 12432]. In the present study, we identified a novel member of the Slp family, Slp5. The same as other Slp family members, the SHD of Slp5 preferentially interacted with the GTP-bound form of Rab27A and marginally with Rab3A and Rab6A, both in vitro and in intact cells, but not with other Rabs tested (Rab1, Rab2, Rab4A, Rab5A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). However, unlike other members of the Slp family, expression of Slp5 mRNA was highly restricted to human placenta and liver. Expression of Slp5 protein and in vivo association of Slp5 with Rab27A in the mouse liver were further confirmed by immunoprecipitation. The results suggest that Slp5 might be involved in Rab27A-dependent membrane trafficking in specific tissues.

Amino Acid Sequence↗
PubMed2002

Invasion of melanoma in double knockout mice lacking tenascin-X and tenascin-C.

The roles of extracellular matrix glycoproteins belonging to the tenascin family in the regulation of tumor cell proliferation, invasion, and metastasis are not known. To address this issue, we generated tenascin-X (TNX) and tenascin-C (TNC) double knockout mice and compared findings in these mice with those in single knockout (TNX + / + TNC - / - and TNX - / - TNC + / +) mice. We investigated the proliferation and invasion of B16-BL6 melanoma cells after these cells had been injected into the footpads of mice in each group. The primary tumor size and invasion to the ankle adjacent to the primary tumor site were examined at 35 days after injection of the melanoma cells. The primary tumor size in TNX - / - TNC + / + mice was significantly larger than that in wild-type mice, but those of TNX + / + TNC - / - and double knockout mice were comparable to that in the wild-type mice. On the other hand, invasion to the ankle was obviously promoted in TNX - / - TNC + / + and double knockout mice compared with that in the wild-type mice, but invasion to the ankle in TNX + / + TNC - / - mice was only slightly promoted. Gelatin zymography confirmed increased matrix metalloproteinase (MMP)-9 activity in the dorsal skin of TNX - / - TNC + / +, TNX + / + TNC - / - and double knockout mice. However, the amounts of MMP-9 mRNA in the dorsal skins of all mice were almost the same, indicating that the increased activity of MMP-9 in the single and double knockout mice is regulated at the MMP-9 processing level. These results indicate that MMP-9 is activated in all TN-deficient mice, but that TNX exerts a greater effect on tumor invasion than does TNC.

Animals↗
PubMed2002

Adhesive defect in extracellular matrix tenascin-X-null fibroblasts: a possible mechanism of tumor invasion.

Extracellular matrix tenascin-X (TNX)-null mice, generated by disruption of the Tnx gene, display augmented invasion and metastasis of B16-BL6 melanoma tumor cells due to increased activities of matrix metalloproteinase (MMP)-2 and MMP-9. In this study, we investigated cell-matrix and cell-cell adhesions using TNX-null fibroblasts and wild-type fibroblasts. TNX-null fibroblasts exhibited a decreased attachment to fibronectin compared with that of wild-type fibroblasts. B16 melanoma cells were cocultured with wild-type or TNX-null fibroblasts, and the adhesion of B16 melanoma to the fibroblasts was assessed. B16 melanoma cells on wild-type fibroblasts proliferated and spread out in a horizontal direction, whereas those on TNX-null fibroblasts overlapped each other rather than migrating horizontally. These overlapping B16 melanoma cells on TNX-null fibroblasts peeled off faster than those on wild-type fibroblasts. To determine whether the decreased cell-matrix and cell-cell adhesions on TNX-null fibroblasts were due to increased MMP activity, the activities of MMPs in wild-type and TNX-null fibroblasts were compared by gelatinolytic assays. The analysis of MMPs from conditioned media demonstrated that almost the same levels of MMP activities were detected between wild-type and TNX-null fibroblasts. However, contrary to our expectations the activities of MMPs from conditioned media of B16 melanoma cells cocultured on TNX-null fibroblasts were rather reduced than those of B16 melanoma cells cocultured on wild-type. We concluded that the absence of TNX in the extracellular environment might play an important role in enhancement of the detachment of B16 melanoma cells.

Animals↗
PubMed2002

DJ-1, a target protein for an endocrine disrupter, participates in the fertilization in mice.

DJ-1 was first identified as an activated ras-dependent oncogene product and was later also found to be an infertility-related protein affected by sperm toxicants such as ornidazole (OR) and epichlorohydrin. These findings suggest that DJ-1 has functions in both somatic cells and sperm. In this study, to determine the relationship between DJ-1 and an endocrine disrupter and to determine the functions of DJ-1 in sperm, in vitro fertilization experiments were carried out using eggs and sperm extracted from mice that had or had not been treated with OR. We found that the amount of DJ-1 in sperm and the efficiency of fertilization decreased with the increasing dose of OR to which the mice were exposed. The addition of an anti-mouse DJ-1 serum to sperm solution before the in vitro fertilization reaction with eggs resulted in a decrease in the efficiency of fertilization to about one-third of that when pre-immune serum was added to sperm solution, indicating that DJ-1 participates in the fertilization.

Animals↗