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Shigeki Shibahara - One of the best experts on this subject based on the ideXlab platform.
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dynamic regulation of the human Dopachrome Tautomerase promoter by mitf er α and chromatin remodelers during proliferation and senescence of human melanocytes
Pigment Cell Research, 2005Co-Authors: Denise J. Schwahn, Shigeki Shibahara, Nikolai A. Timchenko, Estela E. MedranoAbstract:Senescent cells are known to display altered gene expression of differentiation-associated genes. We have previously demonstrated that the melanocyte transcriptional regulator microphthalmia-associated protein (MITF) is down-regulated in senescent melanocytes. Since virtually nothing is known regarding the differentiated function of senescent melanocytes, we analyzed the transcriptional regulation of Dopachrome Tautomerase (DCT), a member of the tyrosinase gene family, in proliferating and in senescent human melanocytes. Computational analysis of the region containing the M-box that includes the MITF CATGTG binding motif demonstrated that this sequence overlaps with the estrogen receptor alpha (ER-alpha), USF-1, TFE-3, Isl-1 and AP-1 binding elements. Electrophoresis gel-shift analysis using an oligonucleotide containing MITF and ERE elements identified MITF and ER-alpha complexes in proliferating melanocytes, whereas only ER-alpha complexes were detected in senescent cells. Importantly, a promoter-reporter analysis demonstrated that the coactivator p300/CBP switched MITF from a repressor to an activator of DCT transcription. p300/CBP was also required by ER-alpha and MITF to induce high, synergistic activation of the DCT promoter. We have also found that transcription of the DCT gene is differentially regulated by major melanocyte mitogens. In contrast to the activating effect of cAMP inducers, 12-O-tetradecanoylphorbolacetate (TPA) was a potent repressor of DCT transcription, suggesting that this gene can be differentially regulated by multiple environmental signals and promoter context. In support of this conclusion, trichostatin A, a histone deacetylase inhibitor, counteracted the TPA-mediated repression, and restored high levels of DCT protein in cultured melanocytes. We conclude that senescent melanocytes display dramatic changes in the expression of differentiation-related proteins; such changes may in turn result in altered melanocyte function and survival to environmental stresses.
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Dynamic regulation of the human Dopachrome Tautomerase promoter by MITF, ER‐α and chromatin remodelers during proliferation and senescence of human melanocytes
Pigment cell research, 2005Co-Authors: Denise J. Schwahn, Shigeki Shibahara, Nikolai A. Timchenko, Estela E. MedranoAbstract:Senescent cells are known to display altered gene expression of differentiation-associated genes. We have previously demonstrated that the melanocyte transcriptional regulator microphthalmia-associated protein (MITF) is down-regulated in senescent melanocytes. Since virtually nothing is known regarding the differentiated function of senescent melanocytes, we analyzed the transcriptional regulation of Dopachrome Tautomerase (DCT), a member of the tyrosinase gene family, in proliferating and in senescent human melanocytes. Computational analysis of the region containing the M-box that includes the MITF CATGTG binding motif demonstrated that this sequence overlaps with the estrogen receptor alpha (ER-alpha), USF-1, TFE-3, Isl-1 and AP-1 binding elements. Electrophoresis gel-shift analysis using an oligonucleotide containing MITF and ERE elements identified MITF and ER-alpha complexes in proliferating melanocytes, whereas only ER-alpha complexes were detected in senescent cells. Importantly, a promoter-reporter analysis demonstrated that the coactivator p300/CBP switched MITF from a repressor to an activator of DCT transcription. p300/CBP was also required by ER-alpha and MITF to induce high, synergistic activation of the DCT promoter. We have also found that transcription of the DCT gene is differentially regulated by major melanocyte mitogens. In contrast to the activating effect of cAMP inducers, 12-O-tetradecanoylphorbolacetate (TPA) was a potent repressor of DCT transcription, suggesting that this gene can be differentially regulated by multiple environmental signals and promoter context. In support of this conclusion, trichostatin A, a histone deacetylase inhibitor, counteracted the TPA-mediated repression, and restored high levels of DCT protein in cultured melanocytes. We conclude that senescent melanocytes display dramatic changes in the expression of differentiation-related proteins; such changes may in turn result in altered melanocyte function and survival to environmental stresses.
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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.
Francisco Solano - One of the best experts on this subject based on the ideXlab platform.
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mutations in Dopachrome Tautomerase dct affect eumelanin pheomelanin synthesis but do not affect intracellular trafficking of the mutant protein
Biochemical Journal, 2005Co-Authors: Gertrudee Costin, Francisco Solano, Kazumasa Wakamatsu, Adina L. Milac, Julio C Valencia, Shosuke Ito, Wilfred D Vieira, Yuji Yamaguchi, Francois Rouzaud, Andrei J PetrescuAbstract:Dopachrome Tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and in the rapid metabolism of its toxic intermediates. Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn congenic C57BL/6J non-agouti black mice and were used to study the effect(s) of these mutations on the intracellular trafficking of Dct and on the pigmentation of the cells. Dct activity is 3-fold lower in slaty cells compared with non-agouti black melanocytes, whereas slaty light melanocytes have a surprisingly 28-fold lower Dct activity. Homology modelling of the active site of Dct suggests that the slaty mutation [R194Q (Arg194→Gln)] is located in the active site and may alter the ability of the enzyme to transform the substrate. Transmembrane prediction methods indicate that the slaty light mutation [G486R (Gly486→Arg)] may result in the sliding of the transmembrane domain towards the N-terminus, thus interfering with Dct function. Chemical analysis showed that both Dct mutations increase pheomelanin and reduce eumelanin produced by melanocytes in culture. Thus the enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis.
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Mutations in Dopachrome Tautomerase (Dct) affect eumelanin/pheomelanin synthesis, but do not affect intracellular trafficking of the mutant protein
Biochemical Journal, 2005Co-Authors: Gertrudee Costin, Francisco Solano, Kazumasa Wakamatsu, Adina L. Milac, Julio C Valencia, Shosuke Ito, Wilfred D Vieira, Yuji Yamaguchi, Francois Rouzaud, Andrei J PetrescuAbstract:Dopachrome Tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and in the rapid metabolism of its toxic intermediates. Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn congenic C57BL/6J non-agouti black mice and were used to study the effect(s) of these mutations on the intracellular trafficking of Dct and on the pigmentation of the cells. Dct activity is 3-fold lower in slaty cells compared with non-agouti black melanocytes, whereas slaty light melanocytes have a surprisingly 28-fold lower Dct activity. Homology modelling of the active site of Dct suggests that the slaty mutation [R194Q (Arg194→Gln)] is located in the active site and may alter the ability of the enzyme to transform the substrate. Transmembrane prediction methods indicate that the slaty light mutation [G486R (Gly486→Arg)] may result in the sliding of the transmembrane domain towards the N-terminus, thus interfering with Dct function. Chemical analysis showed that both Dct mutations increase pheomelanin and reduce eumelanin produced by melanocytes in culture. Thus the enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis.
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Biosynthesis of Neuromelanin and Melanin: The Potential Involvement of Macrophage Inhibitory Factor and Dopachrome Tautomerase as Rescue Enzymes
Catecholamine Research, 2002Co-Authors: Jun Matsunaga, Francisco Solano, Patrick A. Riley, Vincent J. HearingAbstract:This chapter summarizes our recent studies on the regulation of melanin synthesis, a chemical pathway which bears a striking resemblance to that proposed to be involved in neuromelanin production from Dopamine (DN).1 Two key enzymes, Dopachrome Tautomerase (DCT) and macrophage migration inhibitory factor (MIF), have analogous catalytic actions in those biosynthetic pathways which may be linked to their roles as survival enzymes in melanocytes and in catecholaminergic neurons, respectively.
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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.
Eriko Nakamura - One of the best experts on this subject based on the ideXlab platform.
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increase of pro opiomelanocortin mrna prior to tyrosinase tyrosinase related protein 1 Dopachrome Tautomerase pmel 17 gp100 and p protein mrna in human skin after ultraviolet b irradiation
Journal of Investigative Dermatology, 2002Co-Authors: Itaru Suzuki, Tomomi Kato, Tomonori Motokawa, Takayuki Katagiri, Yasushi Tomita, Eriko NakamuraAbstract:In ultraviolet-induced tanning, the protein levels of various gene products critical for pigmentation (including tyrosinase and tyrosinase-related protein-1) are increased in response to ultraviolet B irradiation, but changes in mRNA levels of these factors have not been investigated in vivo . We have established an in situ hybridization technique to investigate mRNA levels of pro-opiomelanocortin, tyrosinase, tyrosinase-related protein-1, Dopachrome Tautomerase, P-protein, Pmel-17/gp100, and microphthalmia-associated transcription factor, and have analyzed the changes in mRNA levels in the ultraviolet B-exposed skin in vivo . The right or left forearm of each volunteer was irradiated with ultraviolet B, and skin biopsies were obtained at 2 and 5 d postirradiation. mRNA level of pro- opiomelanocortin was increased 2 d after ultraviolet B irradiation, and returned to a near-basal level after 5 d, whereas the mRNA levels of tyrosinase, tyrosinase-related protein-1, Dopachrome Tautomerase, P-protein, and Pmel-17/gp100 showed some or no increase at 2 d, but were significantly increased 5 d after ultraviolet B irradiation. Microphthalmia-associated transcription factor mRNA was slightly increased on days 2 and 5 after ultraviolet B irradiation. Our results suggest that the mechanism of the tanning response of human skin may involve the transcriptional regulation of certain pigmentary genes, and that pro-opiomelanocortin-derived melanocortins such as α-melanocyte-stimulating hormone and adrenocorticotropic hormone may play a part in regulating these genes in vivo .
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Increase of pro-opiomelanocortin mRNA prior to tyrosinase, tyrosinase-related protein 1, Dopachrome Tautomerase, Pmel-17/gp100, and P-protein mRNA in human skin after ultraviolet B irradiation.
The Journal of investigative dermatology, 2002Co-Authors: Itaru Suzuki, Tomomi Kato, Tomonori Motokawa, Takayuki Katagiri, Yasushi Tomita, Eriko NakamuraAbstract:In ultraviolet-induced tanning, the protein levels of various gene products critical for pigmentation (including tyrosinase and tyrosinase-related protein-1) are increased in response to ultraviolet B irradiation, but changes in mRNA levels of these factors have not been investigated in vivo . We have established an in situ hybridization technique to investigate mRNA levels of pro-opiomelanocortin, tyrosinase, tyrosinase-related protein-1, Dopachrome Tautomerase, P-protein, Pmel-17/gp100, and microphthalmia-associated transcription factor, and have analyzed the changes in mRNA levels in the ultraviolet B-exposed skin in vivo . The right or left forearm of each volunteer was irradiated with ultraviolet B, and skin biopsies were obtained at 2 and 5 d postirradiation. mRNA level of pro- opiomelanocortin was increased 2 d after ultraviolet B irradiation, and returned to a near-basal level after 5 d, whereas the mRNA levels of tyrosinase, tyrosinase-related protein-1, Dopachrome Tautomerase, P-protein, and Pmel-17/gp100 showed some or no increase at 2 d, but were significantly increased 5 d after ultraviolet B irradiation. Microphthalmia-associated transcription factor mRNA was slightly increased on days 2 and 5 after ultraviolet B irradiation. Our results suggest that the mechanism of the tanning response of human skin may involve the transcriptional regulation of certain pigmentary genes, and that pro-opiomelanocortin-derived melanocortins such as α-melanocyte-stimulating hormone and adrenocorticotropic hormone may play a part in regulating these genes in vivo .
Yasushi Tomita - One of the best experts on this subject based on the ideXlab platform.
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increase of pro opiomelanocortin mrna prior to tyrosinase tyrosinase related protein 1 Dopachrome Tautomerase pmel 17 gp100 and p protein mrna in human skin after ultraviolet b irradiation
Journal of Investigative Dermatology, 2002Co-Authors: Itaru Suzuki, Tomomi Kato, Tomonori Motokawa, Takayuki Katagiri, Yasushi Tomita, Eriko NakamuraAbstract:In ultraviolet-induced tanning, the protein levels of various gene products critical for pigmentation (including tyrosinase and tyrosinase-related protein-1) are increased in response to ultraviolet B irradiation, but changes in mRNA levels of these factors have not been investigated in vivo . We have established an in situ hybridization technique to investigate mRNA levels of pro-opiomelanocortin, tyrosinase, tyrosinase-related protein-1, Dopachrome Tautomerase, P-protein, Pmel-17/gp100, and microphthalmia-associated transcription factor, and have analyzed the changes in mRNA levels in the ultraviolet B-exposed skin in vivo . The right or left forearm of each volunteer was irradiated with ultraviolet B, and skin biopsies were obtained at 2 and 5 d postirradiation. mRNA level of pro- opiomelanocortin was increased 2 d after ultraviolet B irradiation, and returned to a near-basal level after 5 d, whereas the mRNA levels of tyrosinase, tyrosinase-related protein-1, Dopachrome Tautomerase, P-protein, and Pmel-17/gp100 showed some or no increase at 2 d, but were significantly increased 5 d after ultraviolet B irradiation. Microphthalmia-associated transcription factor mRNA was slightly increased on days 2 and 5 after ultraviolet B irradiation. Our results suggest that the mechanism of the tanning response of human skin may involve the transcriptional regulation of certain pigmentary genes, and that pro-opiomelanocortin-derived melanocortins such as α-melanocyte-stimulating hormone and adrenocorticotropic hormone may play a part in regulating these genes in vivo .
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Increase of pro-opiomelanocortin mRNA prior to tyrosinase, tyrosinase-related protein 1, Dopachrome Tautomerase, Pmel-17/gp100, and P-protein mRNA in human skin after ultraviolet B irradiation.
The Journal of investigative dermatology, 2002Co-Authors: Itaru Suzuki, Tomomi Kato, Tomonori Motokawa, Takayuki Katagiri, Yasushi Tomita, Eriko NakamuraAbstract:In ultraviolet-induced tanning, the protein levels of various gene products critical for pigmentation (including tyrosinase and tyrosinase-related protein-1) are increased in response to ultraviolet B irradiation, but changes in mRNA levels of these factors have not been investigated in vivo . We have established an in situ hybridization technique to investigate mRNA levels of pro-opiomelanocortin, tyrosinase, tyrosinase-related protein-1, Dopachrome Tautomerase, P-protein, Pmel-17/gp100, and microphthalmia-associated transcription factor, and have analyzed the changes in mRNA levels in the ultraviolet B-exposed skin in vivo . The right or left forearm of each volunteer was irradiated with ultraviolet B, and skin biopsies were obtained at 2 and 5 d postirradiation. mRNA level of pro- opiomelanocortin was increased 2 d after ultraviolet B irradiation, and returned to a near-basal level after 5 d, whereas the mRNA levels of tyrosinase, tyrosinase-related protein-1, Dopachrome Tautomerase, P-protein, and Pmel-17/gp100 showed some or no increase at 2 d, but were significantly increased 5 d after ultraviolet B irradiation. Microphthalmia-associated transcription factor mRNA was slightly increased on days 2 and 5 after ultraviolet B irradiation. Our results suggest that the mechanism of the tanning response of human skin may involve the transcriptional regulation of certain pigmentary genes, and that pro-opiomelanocortin-derived melanocortins such as α-melanocyte-stimulating hormone and adrenocorticotropic hormone may play a part in regulating these genes in vivo .
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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.
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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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Preparation of purified tyrosinase devoid of Dopachrome Tautomerase from mammalian malignant melanocytes.
Pigment cell research, 1993Co-Authors: Paloma Valverde, Josè Carlos Garcia-borron, Francisco Solano, Celia Jiménez-cervantes, C. Salinas, Joséa. LozanoAbstract:Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of Dopachrome Tautomerase (DCT2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60 degrees C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.
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effect of amphotericin b on Dopachrome Tautomerase activity and other melanogenic parameters in cultured b16 f10 melanoma cells
Pigment Cell Research, 1992Co-Authors: Paloma Peinado, J H Martinezliarte, Francisco Solano, Joséa. LozanoAbstract:The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in Dopachrome Tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.