The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Shigeki Shibahara - One of the best experts on this subject based on the ideXlab platform.
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OTX2 regulates expression of Dopachrome tautomerase in human retinal pigment epithelium.
Biochemical and biophysical research communications, 2003Co-Authors: Kazuhisa Takeda, Ken-ichi Yasumoto, Tetsuo Udono, Kazuhiro Takahashi, Satoru Yokoyama, Hideo Saito, Shigeki ShibaharaAbstract:Otx2 is a member of homeodomain-containing transcription factors and is essential for eye morphogenesis in mice. Here we show the expression of OTX2, the human counterpart of Otx2, in cell lines of retinal pigment epithelium (RPE) and in Y79 retinoblastoma cells that exhibit the property of presumptive RPE. These RPE cells express Dopachrome tautomerase (DCT) that is an enzyme involved in melanin biosynthesis. DCT may contribute to the homeostasis of RPE by detoxifying DOPA-derived metabolites. OTX2 binds to the DCT gene promoter in vivo, as judged by chromatin immunoprecipitation assays. Furthermore, repression of endogenous OTX2 expression in Y79 cells by an anti-sense OTX2 oligonucleotide resulted in the decrease of DCT protein contents. Transient expression assays revealed that OTX2 activated the DCT gene promoter through the OTX-2-binding site in an RPE-specific manner. Therefore, OTX2 may regulate RPE-specific target genes, such as DCT, thereby maintaining the homeostasis of RPE.
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Expression of tyrosinase-related protein 2/Dopachrome tautomerase in the retinoblastoma.
Experimental eye research, 2001Co-Authors: Tetsuo Udono, Ken-ichi Yasumoto, Kazuhiro Takahashi, Miki Yoshizawa, Kazuhisa Takeda, Toshiaki Abe, Makoto Tamai, Shigeki ShibaharaAbstract:Tyrosinase-related protein 2 (TRP-2), also known as Dopachrome tautomerase, is an enzyme in melanin biosynthesis and may play an important role in detoxification of a metabolite derived from DOPA. TRP-2 is expressed in melanocytes of neural crest origin and retinal pigment epithelium (RPE), derived from the optic cup. TRP-2 has been established as an early differentiation marker for melanoblasts and RPE. It is therefore of significance to study the regulation of TRP-2/Dopachrome tautomerase expression. Here we show that TRP-2 mRNA is expressed in Y79 human retinoblastoma cell line, derived from a primitive multipotential retinal cell. Retinoblastoma is the common primary intraocular tumor of childhood. Basal expression levels in Y79 retinoblastoma cells of TRP-2 mRNA and protein are comparable to those in melanoma cells, whereas mRNA for tyrosinase, the rate-limiting enzyme in melanogenesis, is undetectable in retinoblastoma cells. Transient transfection assays showed that the TRP-2 gene promoter efficiently directs the reporter gene expression in retinoblastoma cells as it does in melanoma cells. Moreover, the expression of TRP-2 mRNA was induced by retinoic acid in retinoblastoma cells but not noticeably affected by forskolin, a cAMP-elevating reagent, whereas in melanoma cells its expression was induced by forskolin but not by retinoic acid. These results suggest a difference in the regulation of TRP-2 expression between retinoblastoma and melanoma cells. Moreover, TRP-2 mRNA is expressed in the excised retinoblastoma specimens, as assessed by RT-PCR. The present study shows unexpected features of TRP-2 and may enhance our understanding of the pathophysiology of retinoblastoma.
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Identification of a composite enhancer of the human tyrosinase-related protein 2/Dopachrome tautomerase gene.
Biochimica et Biophysica Acta, 2000Co-Authors: Shintaro Amae, Ken-ichi Yasumoto, Tetsuo Udono, Kazuhiro Takahashi, Kazuhisa Takeda, Shigeki ShibaharaAbstract:Abstract The human tyrosinase-related protein 2 (TRP-2) gene promoter contains a cis -regulatory element (positions −447 to −416), termed Dopachrome tautomerase distal enhancer 1 (DDE1). DDE1 functions as an enhancer in cultured melanoma cells and its core element includes a potential binding site for transcription factors containing a high-mobility-group domain. This core element is bound in vitro by multiple nuclear proteins, which are preferentially expressed in melanoma cells. DDE1 represents a composite enhancer that may be involved in melanocyte-specific transcription of the human TRP-2 gene.
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molecular cloning and functional analysis of a cdna coding for human Dopachrome tautomerase tyrosinase related protein 2
Biochimica et Biophysica Acta, 1994Co-Authors: Kouji Yokoyama, Yasushi Tomita, Hiroyuki Suzuki, Ken-ichi Yasumoto, Shigeki ShibaharaAbstract:We have cloned the cDNAs encoding tyrosinase-related protein-2 (TRP-2) from a human melanoma cDNA library. Transient expression of the isolated cDNA in HeLa cells established that TRP-2 is Dopachrome tautomerase. Human TRP-2/Dopachrome tautomerase is composed of 519 amino acids with a molecular weight of 59,000 and has about 84% identity with the mouse counterpart.
Joséa. Lozano - One of the best experts on this subject based on the ideXlab platform.
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Molecular mechanism for catalysis by a new zinc-enzyme, Dopachrome tautomerase.
Biochemical Journal, 1996Co-Authors: Francisco Solano, Josè Carlos Garcia-borron, J H Martinez-liarte, Jose R. Jara, Celia Jiménez-cervantes, Joséa. LozanoAbstract:Dopachrome tautomerase (DCT; EC 5.3.3.12) catalyses the conversion of L-Dopachrome into 5,6-dihydroxyindole-2-carboxylic acid in the mammalian eumelanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to a family of three metalloenzymes termed the tyrosinase-related proteins (TRPs). It is well known that tyrosinase has copper in its active site. However, the nature of the metal ion in the active site of DCT is under discussion. Whereas theoretical predictions based on similarity between the protein sequences of the TRPs suggest the presence of copper, the different inhibition pattern of DCT with some metal chelators compared with that of tyrosinase suggests that the nature of the metal ion could differ. Direct estimations of the metal content in purified DCT preparations show the presence of around 1.5 Zn atoms/molecule and the absence of copper. Apoenzyme preparation by treatment of DCT with cyanide or o-phenanthroline followed by reconstitution experiments of tautomerase activity in the presence of different ions confirmed that the metal cofactor for the DCT active site is zinc. Our results are consistent with Zn2+ chelation by the highly conserved histidine residues homologous to the histidines at the classical copper-binding sites in tyrosinase. This finding accounts for the reaction catalysed by DCT, i.e. a tautomerization, versus the copper-mediated oxidations catalysed by tyrosinase. Based on the predicted tetrahedrical coordination of the zinc ions in the enzyme active site, a molecular mechanism for the catalysis of L-Dopachrome tautomerization is proposed. From the present data, the existence of additional ligands for metal ions other than zinc in the DCT molecule, such as the proposed cysteine iron-binding sites, cannot be completely ruled out. However, if such sites exist, they could be subsidiary binding sites, whose function would be likely to stabilize the protein.
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Dopachrome tautomerase is a zinc-containing enzyme.
Biochemical and biophysical research communications, 1994Co-Authors: Francisco Solano, Josè Carlos Garcia-borron, J H Martinez-liarte, Celia Jiménez-cervantes, Joséa. LozanoAbstract:Abstract Dopachrome tautomerase (DCT) catalyzes the conversion of L-Dopachrome into 5,6-dihydroxyindole-2-carboxylic acid through the melanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to the family of the tyrosinase related proteins. The three members of the family contain two highly conserved metal-binding sites with three histidines on each. Tyrosinase has copper at its active site. If was assumed that although DCT might have copper in those metal binding sites, its active site could be related to other two putative iron-binding sites located in different positions. Based on apoDCT preparation with cyanide and reconstitution experiments, we propose that DCT have zinc instead of copper at the two metal-binding sites and that those sites actually correspond to the active site. The involvement of zinc, which cannot undergo redox reactions, accounts for the reaction that DCT catalyzes, a tautomerization versus the copper-mediated oxidations catalyzed by tyrosinase.
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Dopachrome tautomerase decreases the binding of indolic melanogenesis intermediates to proteins
Biochimica et biophysica acta, 1994Co-Authors: Cristina Salinas, Francisco Solano, Josè Carlos Garcia-borron, Joséa. LozanoAbstract:Abstract Dopachrome tautomerase (DCT) is a recently characterized enzyme contributing to the control of melanogenesis in mammals. The enzyme catalyzes the rearrangement of l -Dopachrome ( l -DC) to 5,6-dihydroxyindole 2-carboxylic acid (DHICA), while the spontaneous rearrangement of l -DC leads to 5,6-dihydroxyindole (DHI). Due to the lower reactivity of DHICA in comparison to DHI, DCT could provide a protective mechanism against the cytotoxicity of decarboxylated indolic melanogenic intermediates by limiting the formation of these highly reactive decarboxylated species within melanocytes. We have followed the binding of radioactive melanogenic precursors to a model protein, bovine serum albumin (BSA). Using l -DC as initial melanin precursor, this binding was decreased by DCT in a concentration-dependent manner. In the presence of tyrosinase, the binding of l -DOPA-derived intermediates to BSA was also decreased by DCT and the percentage of decrease was even higher than using l -DC as initial melanin precursor. SDS-PAGE followed by fluorographic detection of radioactive bands showed the formation of covalent adducts between BSA and melanin precursors, as well as of aggregated forms of this protein. This aggregation was also diminished by DCT. These data indicate that DCT could play a protective role against the cytotoxic action of decarboxylated indoles within mammalian melanocytes.
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α msh and other melanogenic activators mediate opposite effects of tyrosinase and Dopachrome tautomerase in b16 f10 mouse melanoma cells
Journal of Investigative Dermatology, 1992Co-Authors: J H Martinezliarte, Jose R. Jara, Francisco Solano, Jose C Garciaborron, Joséa. LozanoAbstract:Abstract α-MSH was found to decrease the recently characterized Dopachrome tautomerase activity in cultures of B16/F10 mouse melanoma cells. Other stimulating agents of melanogenesis, like dibutyryl cyclic AMP, 3-isobutyl-l-methylxanthine, theophylline, retinol, and retinoic acid, caused the same effect. The grade of inhibition depended on the nature of the agent and the time of exposure. In all cases, both melanin production and tyrosinase activity were activated by these treatments, although the grade of tyrosine hydroxylase and dopa oxidase stimulation was different. Moreover, no correlation among the intensities of Dopachrome tautomerase inhibition and tyrosinase activation by the tested agents could be obtained. The significance of these results in the regulation of mammalian melanogenesis is discussed.
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Regulation of the final phase of mammalian melanogenesis. The role of Dopachrome tautomerase and the ratio between 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole.
European journal of biochemistry, 1992Co-Authors: Pilar Aroca, Josè Carlos Garcia-borron, Francisco Solano, Cristina Salina, Joséa. LozanoAbstract:The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (Dopachrome) formation, is studied. It is shown that both tyrosinase and Dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas Dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of Dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-Dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-Dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of Dopachrome tautomerase and tyrosinase.
Friedrich Beermann - One of the best experts on this subject based on the ideXlab platform.
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Regulation of DHICA-mediated antioxidation by Dopachrome tautomerase: implication for skin photoprotection against UVA radiation.
Free radical biology & medicine, 2010Co-Authors: Shan Jiang, Friedrich Beermann, Xiao-ming Liu, Xin Dai, Qiong Zhou, Tie-chi Lei, Kazumasa WakamatsuAbstract:Dopachrome tautomerase (Dct) is a critical enzyme in the melanogenesis pathway that isomerizes the intermediate Dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and influences the proportion of DHICA monomer incorporated into the 5,6-dihydroxyindole (DHI) polymer in eumelanin. To investigate whether Dct inactivation affects skin photoprotection against ultraviolet radiation, we examined levels of reactive oxygen species (ROS), sunburn cell formation, epidermal cell apoptosis, and melanin composition in skins of Dct-/-knockout mice compared with skins of wild-type C57 BL/6 mice under UVA-induced oxidative stress. The results demonstrate that Dct inactivation elevates the level of ROS, increases the numbers of sunburn cells and apoptotic cells, decreases the amount of eumelanin in the epidermis upon exposure to chronic UVA radiation. Moreover, we determined the effects of DHICA-melanin, DHI-melanin, and a mixture of both on hydroxyl radical generation in the Fenton reaction utilizing an electron spin resonance assay. DHICA-melanin exhibits a potent hydroxyl radical scavenging activity, whereas DHI-melanin does not. Thus, this study suggests that DHICA monomers are required to incorporate into the DHI polymer backbone of eumelanin, which highlights the important role of Dct played in the regulation of DHICA-mediated antioxidation
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Melanocytes and Pigmentation Are Affected in Dopachrome Tautomerase Knockout Mice
Molecular and cellular biology, 2004Co-Authors: Laurence Guyonneau, Fabien Murisier, Anita Rossier, Alexandre Moulin, Friedrich BeermannAbstract:The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and Dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dct(tm1(Cre)Bee)] lack Dct mRNA and Dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dct(slt)/Dct(slt)) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.
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tyrosinase and related proteins in mammalian pigmentation
FEBS Letters, 1996Co-Authors: Veronique Del Marmol, Friedrich BeermannAbstract:Tyrosinase is the key enzyme in pigment synthesis, initiating a cascade of reactions which convert the amino acid tyrosine to the melanin biopolymer. Two other tyrosinase-related proteins (TRP) are known, TRP-1 (probably DHICAoxidase) and TRP-2 (Dopachrome tautomerase). These proteins show about 40% homology, and recent results have indicated that the genes might be derived from a common ancestor. We will discuss recent findings on genomic organization, and on the proteins and their presumed function, which is important for eumelanin synthesis in mouse and man.
Jun Nishihira - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of human D-Dopachrome tautomerase, a homologue of macrophage migration inhibitory factor, at 1.54 A resolution.
Biochemistry, 1999Co-Authors: Hiroshi Sugimoto, Masaki Suzuki, Isao Tanaka, Masae Taniguchi, Atsushi Nakagawa, Jun NishihiraAbstract:D-Dopachrome tautomerase shares a low homologous amino acid sequence (33% homology) with the macrophage migration inhibitory factor (MIF) and possesses similar tautomerase activity as well. MIF is a cytokine involved in inflammatory reactions and immune responses. Whereas recent studies have identified MIF as a pituitary hormone and immunoregulator, much less is known about the structural basis of these physiological functions and the real significance of tautomerase activity. Therefore, interest in the structure-function relationship between D-Dopachrome tautomerase and MIF has increased, especially with regard to inflammation and immune responses. We have determined the X-ray crystal structure of human D-Dopachrome tautomerase at 1.54 A resolution. D-Dopachrome tautomerase folds to form a homotrimer that has extensive contact between subunits by intersubunit beta-sheets. Its overall topology and trimeric formations are similar to those of human MIF. The N-terminal proline is located at the bottom of a positively charged pocket in which the conformations of Lys32 and Ser63 are highly conserved. These positively charged properties are also seen in the active site pocket of human MIF, bacterial 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and 4-oxalocrotonate tautomerase (4-OT). A detailed comparison of these structures revealed significant differences in the environment around the potential active site, the intersubunit contacts, and charge distribution on the molecular surface. It can be concluded that these features are related to the physiological role and tautomerase activity of MIF and D-Dopachrome tautomerase. The present structural study could be helpful for designing effective inhibitors that modulate immunoregulatory and hormone-like effects.
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Cloning of the mouse gene for D-Dopachrome tautomerase.
Biochimica et biophysica acta, 1998Co-Authors: Takeshi Kuriyama, Mami Fujinaga, Toshiaki Koda, Jun NishihiraAbstract:D-Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5, 6-quinone (D-Dopachrome) into 5,6-dihydroxyindole. The amino acid sequence of this protein is 27% identical with that of macrophage migration inhibitory factor, which is known as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. In this study, we isolated and sequenced a 3490 bp-long genomic DNA of mouse D-Dopachrome tautomerase that consists of three exons and two introns. By two procedures, 5' rapid amplification of cDNA ends and cap site labeling, we determined the transcription initiation site, which is located 46 bp upstream of the translation initiation site. The possible polyadenylation sequence (AATAAA) is located 180 bp downstream of the termination codon. Computer-assisted analysis of the nucleotide sequence revealed a number of regulatory motifs, including multiple sites for Sp1, C/EBP, NF-Y, and USF. Although the precise pathophysiological functions of D-Dopachrome tautomerase remain to be elucidated, the present results will contribute not only to elucidation of the mechanism of gene expression, but also to understanding of the molecular function of this protein.
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Molecular Cloning of Human D-Dopachrome Tautomerase cDNA: N-terminal Proline Is Essential for Enzyme Activation
Biochemical and biophysical research communications, 1998Co-Authors: Jun Nishihira, Masaki Suzuki, Isao Tanaka, Hiroshi Sugimoto, Takeshi Kuriyama, Mami Fujinaga, Atsushi Nakagawa, Masaharu SakaiAbstract:Abstract d -Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5,6-quinone ( d -Dopachrome) into 5,6-dihydroxyindole. This protein has an amino acid sequence that is highly homologous with that of macrophage migration inhibitory factor (MIF), which has the potential to catalyze d -Dopachrome to 5,6-dihydroxyindole-2-carboxylic acid and is an important cytokine for T-lymphocyte activation. We isolated and sequenced a 566 bp-long cDNA encoding human d -Dopachrome tautomerase. The cDNA contains an open reading frame encoding 118 amino acids, including the initiator methionine. The amino acid sequence of the protein shares 80% homology with that of the rat enzyme. Northern blot analysis demonstrated that mRNA of d -Dopachrome tautomerase is expressed in a large amount in the liver, and to lesser extent in other organs, including the heart, lung and pancreas. After purification of d -Dopachrome tautomerase expressed in E. coli, we confirmed that the recombinant protein catalyzes the conversion of d -Dopachrome to 5,6-dihydroxyindole. Its catalytic mechanism is not well understood. We found that the protein completely lost the enzyme activity when the N-terminal proline residue was replaced with alanine by site-directed mutagenesis. This fact suggests that the N-terminal proline is essential for the catalytic mechanism. Although the precise pathophysiological function of d -Dopachrome tautomerase remains to be elucidated, the present results could contribute to further understanding of isomerase activity in relation to the immune response.
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substrate specificity for isomerase activity of macrophage migration inhibitory factor and its inhibition by indole derivatives
Journal of Biochemistry, 1997Co-Authors: Masaki Suzuki, Isao Tanaka, Hiroshi Sugimoto, Jun NishihiraAbstract:: Macrophage migration inhibitory factor (MIF) was discovered as a cytokine that inhibits random migration of macrophages and concentrates them at inflammatory loci. We recently reported the tertiary structure of MIF, and revealed its similarity to that of 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, MIF was found to have isomerase activity converting D-Dopachrome, a stereoisomer of naturally-occurring L-Dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. In this study, we examined the effects of a series of compounds analogous to D-Dopachrome on the enzyme activity to obtain vital information for identification of a natural substrate of MIF. Adrenochrome, lacking a carboxyl group at position 2 of the indolinequinone ring, could not be a substrate. Several indole-ring-containing compounds with a carboxyl group were inhibitory to D-Dopachrome isomerase activity, of which indole-3-acrylic acid was the most potent inhibitor, with an inhibitor constant (Ki) of 2.8 mM. 2,3-Indolinedione, which lacks a complete indole ring or a carboxyl group but has carbonyl groups at positions 2 and 3, apparently inhibited the enzyme activity in a competitive or mixed manner with a Ki of 0.9 mM. Taken together, these facts suggest that the 2-carboxyl group of the substrate is essential for interaction with the active site of MIF.
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Crystallization and preliminary X-ray analysis of human D-Dopachrome tautomerase.
Journal of structural biology, 1997Co-Authors: Hiroshi Sugimoto, Masaki Suzuki, Isao Tanaka, Masae Taniguchi, Atsushi Nakagawa, Jun NishihiraAbstract:Abstract d -Dopachrome tautomerase catalyzes the conversion of d -Dopachrome to 5,6-dihydroxyindole. This protein has amino acid sequence homology with that of macrophage migration inhibitory factor (MIF), suggesting a pathophysiological role of this protein in inflammatory and immunological events. We previously determined the tertiary structure of MIF and revealed the functional and evolutional relationships of this protein to isomerase. However, the reaction mechanism of both proteins associated with the inflammatory response, immune system, or tautomerase activities in vitro have not yet been clarified. The tertiary structure of d -Dopachrome tautomerase would provide insight into the molecular function and the mechanism of these proteins. In this study, we crystallized human d -Dopachrome tautomerase by a hanging-drop vapor diffusion method. The crystals belong to the trigonal space group P 3, with unit cell dimensions a = b = 84.2 A and c = 41.0 A. They contain three (or two) monomers in the asymmetric unit, corresponding to a V M value of 2.21 (or 3.32) A 3 Da −1 . The best crystals diffract X-ray to 1.6 A resolution using a synchrotron radiation source. Crystallization of the selenomethionyl derivative of the protein for applying the multiwavelength anomalous diffraction method was also successful.
Ken-ichi Yasumoto - One of the best experts on this subject based on the ideXlab platform.
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OTX2 regulates expression of Dopachrome tautomerase in human retinal pigment epithelium.
Biochemical and biophysical research communications, 2003Co-Authors: Kazuhisa Takeda, Ken-ichi Yasumoto, Tetsuo Udono, Kazuhiro Takahashi, Satoru Yokoyama, Hideo Saito, Shigeki ShibaharaAbstract:Otx2 is a member of homeodomain-containing transcription factors and is essential for eye morphogenesis in mice. Here we show the expression of OTX2, the human counterpart of Otx2, in cell lines of retinal pigment epithelium (RPE) and in Y79 retinoblastoma cells that exhibit the property of presumptive RPE. These RPE cells express Dopachrome tautomerase (DCT) that is an enzyme involved in melanin biosynthesis. DCT may contribute to the homeostasis of RPE by detoxifying DOPA-derived metabolites. OTX2 binds to the DCT gene promoter in vivo, as judged by chromatin immunoprecipitation assays. Furthermore, repression of endogenous OTX2 expression in Y79 cells by an anti-sense OTX2 oligonucleotide resulted in the decrease of DCT protein contents. Transient expression assays revealed that OTX2 activated the DCT gene promoter through the OTX-2-binding site in an RPE-specific manner. Therefore, OTX2 may regulate RPE-specific target genes, such as DCT, thereby maintaining the homeostasis of RPE.
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Expression of tyrosinase-related protein 2/Dopachrome tautomerase in the retinoblastoma.
Experimental eye research, 2001Co-Authors: Tetsuo Udono, Ken-ichi Yasumoto, Kazuhiro Takahashi, Miki Yoshizawa, Kazuhisa Takeda, Toshiaki Abe, Makoto Tamai, Shigeki ShibaharaAbstract:Tyrosinase-related protein 2 (TRP-2), also known as Dopachrome tautomerase, is an enzyme in melanin biosynthesis and may play an important role in detoxification of a metabolite derived from DOPA. TRP-2 is expressed in melanocytes of neural crest origin and retinal pigment epithelium (RPE), derived from the optic cup. TRP-2 has been established as an early differentiation marker for melanoblasts and RPE. It is therefore of significance to study the regulation of TRP-2/Dopachrome tautomerase expression. Here we show that TRP-2 mRNA is expressed in Y79 human retinoblastoma cell line, derived from a primitive multipotential retinal cell. Retinoblastoma is the common primary intraocular tumor of childhood. Basal expression levels in Y79 retinoblastoma cells of TRP-2 mRNA and protein are comparable to those in melanoma cells, whereas mRNA for tyrosinase, the rate-limiting enzyme in melanogenesis, is undetectable in retinoblastoma cells. Transient transfection assays showed that the TRP-2 gene promoter efficiently directs the reporter gene expression in retinoblastoma cells as it does in melanoma cells. Moreover, the expression of TRP-2 mRNA was induced by retinoic acid in retinoblastoma cells but not noticeably affected by forskolin, a cAMP-elevating reagent, whereas in melanoma cells its expression was induced by forskolin but not by retinoic acid. These results suggest a difference in the regulation of TRP-2 expression between retinoblastoma and melanoma cells. Moreover, TRP-2 mRNA is expressed in the excised retinoblastoma specimens, as assessed by RT-PCR. The present study shows unexpected features of TRP-2 and may enhance our understanding of the pathophysiology of retinoblastoma.
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Identification of a composite enhancer of the human tyrosinase-related protein 2/Dopachrome tautomerase gene.
Biochimica et Biophysica Acta, 2000Co-Authors: Shintaro Amae, Ken-ichi Yasumoto, Tetsuo Udono, Kazuhiro Takahashi, Kazuhisa Takeda, Shigeki ShibaharaAbstract:Abstract The human tyrosinase-related protein 2 (TRP-2) gene promoter contains a cis -regulatory element (positions −447 to −416), termed Dopachrome tautomerase distal enhancer 1 (DDE1). DDE1 functions as an enhancer in cultured melanoma cells and its core element includes a potential binding site for transcription factors containing a high-mobility-group domain. This core element is bound in vitro by multiple nuclear proteins, which are preferentially expressed in melanoma cells. DDE1 represents a composite enhancer that may be involved in melanocyte-specific transcription of the human TRP-2 gene.
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molecular cloning and functional analysis of a cdna coding for human Dopachrome tautomerase tyrosinase related protein 2
Biochimica et Biophysica Acta, 1994Co-Authors: Kouji Yokoyama, Yasushi Tomita, Hiroyuki Suzuki, Ken-ichi Yasumoto, Shigeki ShibaharaAbstract:We have cloned the cDNAs encoding tyrosinase-related protein-2 (TRP-2) from a human melanoma cDNA library. Transient expression of the isolated cDNA in HeLa cells established that TRP-2 is Dopachrome tautomerase. Human TRP-2/Dopachrome tautomerase is composed of 519 amino acids with a molecular weight of 59,000 and has about 84% identity with the mouse counterpart.