Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.1 (tyrosinase)
9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to better understand the cascade of melanogenic events in melanocytes, this report has introduced our two recent approaches for the expression of melanogenesis/or melanosome-associated genes and encoded proteins in melanocytes (melanoma cells) after repeated exposure to UV-B and after cotransfection of two human genes, i.e., tyrosinase and tyrosinase-related protein-1 (TRP-1). Repeated exposure of UV-B (2.5-5.0 mJ/cm2) caused not only upregulation of tyrosinase and TRP-1 genes but also coordinated increase in the gene and protein synthesis expression of Lamp-1 (lysosome-associated membrane protein-1). When COS-7 kidney cells and amelanotic melanoma (C32 and SK-MEL-24) and melanotic melanoma (G361 and SK-MEL-23) cells were exposed to cotransfection of human tyrosinase and TRP-1 cDNAs, there was also an increased expression of Lamp-1 mRNA and protein along with tyrosinase activation and new melanin synthesis. Importantly, single transfectants of human tyrosinase cDNA revealed marked cellular degeneration, whereas this degeneration was not seen in single transfectants of TRP-1 cDNA or cotransfectants of human tyrosinase and TRP-1 cDNAs, indicating that TRP-1 prevented, along with Lamp-1, programmed death of melanocytes after transfection of tyrosinase gene. The coordinated expression of TRP-1 and Lamp-1 was further confirmed by antisense oligodeoxynucleotide hybridization experiment against Lamp-1 gene, showing the decreased expression of TRP-1 as identified by three different types of anti-TRP-1 monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Coordinated mRNA and protein expression of human LAMP-1 in induction of melanogenesis after UV-B exposure and co-transfection of human tyrosinase and TRP-1 cDNAs. 788 4

Vitiligo is a common depigmenting skin disease, associated with certain autoimmune endocrinopathies, and autoantibodies to several antigens can be found in melanoma cells. We set out to identify the antigens. We examined 26 patients with vitiligo and associated endocrine disease. Of these, 18 patients (77%) and 8 immediate family members had autoantibodies specific for a 69 kDa protein in HTB-70 human melanoma cells that was not seen in control cells. The autoantibody-positive patient sera reacted with recombinant human tyrosinase expressed in Escherichia coli seen by western blots, as did antibodies raised in rabbits against hamster tyrosinase, but not to recombinant tyrosinase-related protein. Not one of 31 normal controls or 8 patients with alopecia or systemic lupus erythematosus had tyrosinase autoantibodies but a small proportion (12%) of 42 patients with autoimmune endocrine disease without a history of vitiligo had them. The results show that tyrosinase, an enzyme important in melanin formation, is a principal autoantigen of autoimmune vitiligo.
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PMID:The role of tyrosinase in autoimmune vitiligo. 793 46

We constructed two genes specific to melanogenesis, human tyrosinase (HT) and tyrosinase-related protein-1 (TRP-1) genes, into two separate expression vectors so that the cloned genes were under the control of a human cytomegalovirus promoter and enhancer. Monkey kidney COS-7 cells and human amelanotic and melanotic melanoma cells were then cotransfected by both HT and TRP-1 or transfected individually with each gene. The transfectants were examined for mRNA expression by reverse transcription-mediated RNA-PCR amplification. HT or TRP-1 mRNA was strongly expressed in HT or TRP-1 transfectants and cotransfectants of the two genes. Both light and electron microscopic observations indicated that degeneration and premature death of melanocytes occurred in HT transfectants, but not in TRP-1 transfectants or in HT and TRP-1 cotransfectants. Cotransfected cells from five cell lines revealed numerous granular reaction products with an anti-TRP-1 antibody and lysosomal granules with electron-dense material. Our melanin assay confirmed the new production of melanin pigments in these cells, indicating that the lysosomal granules would contain melanin pigments. The gene expression studies of lysosomal protein (beta-galactosidase, CD63, Lamp-1, and Lamp-2) revealed a dramatically elevated gene expression of Lamp-1, which is associated with the membrane receptor of lysosomal granules, in HT- and TRP-1-cotransfected cells. Conversely, the treatment of melanoma cells with antisense oligodeoxynucleotides against Lamp-1 resulted in a decreased expression of TRP-1 protein by immunoprecipitation, supporting the observations of the HT and TRP-1 cotransfection study regarding the up-regulation of Lamp-1 expression. We conclude that HT, TRP-1, and Lamp-1 gene products may function together, being expressed as a multiprotein complex within the melanosomal compartment. Specifically, HT and TRP-1 may function together via Lamp-1 by stabilizing the enzyme-protein complex within the melanosome and prevent the premature death of melanocytes due to tyrosinase-mediated cytotoxicity.
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PMID:Cotransfection of genes encoding human tyrosinase and tyrosinase-related protein-1 prevents melanocyte death and enhances melanin pigmentation and gene expression of Lamp-1. 802 May 95

Since the characterization of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as a major melanogenic intermediate, the fate of this compound and the mechanisms of its incorporation into the melanin polymer have become major issues in the study of melanogenesis. DHICA is a stable dihydroxyindole with a low rate of spontaneous oxidation, suggesting that enzymatic mechanism(s) might contribute to its evolution. The most obvious candidates are the melanosomal tyrosinases. We have recently shown that mouse melanosomes contain two electrophoretically distinct tyrosinase isoenzymes, termed low electrophoretic mobility tyrosinase (LEMT) and high electrophoretic mobility tyrosinase (HEMT), that can be resolved and purified. In this study, we report immunological evidence indicating that LEMT corresponds to the protein encoded by the brown locus (termed tyrosinase-related protein-1, TRP1), while HEMT corresponds to the tyrosinase encoded by the albino locus. We have compared the ability of both isoenzymes to catalyze DHICA evolution as determined by high performance liquid chromatography; although LEMT is a relatively poor tyrosine hydroxylase and DOPA oxidase as compared to HEMT, it was readily able to accelerate DHICA consumption concomitant with the production of a brownish product. However, the DHICA conversion activity of HEMT was barely detectable. The ability of purified LEMT to catalyze DHICA conversion could be almost completely abolished by treatment with heat or trypsin, and was inhibited in a concentration dependent way by the tyrosinase inhibitor 2-phenylthiourea and by L-tyrosine. Moreover, in the presence of low concentration of ascorbate, the DHICA conversion activity of LEMT displayed a lag period which was progressively longer at higher ascorbate concentrations. Based on the relationship between ascorbate added, enzyme activity, and lag period, it is very likely that the DHICA converting activity is indeed a DHICA oxidase activity. This was further proven by the demonstration that the product reacts rapidly and efficiently with the quinone trapping reagent 3-methyl-2-benzothiazolinone hydrazone, yielding a colored adduct similar to the one obtained with DOPAquinone. The DHICA oxidase activity of LEMT displayed a Km for DHICA of about 0.8 mM, as compared to 1.9 mM for L-DOPA and 0.23 nM for L-tyrosine. These results suggest that TRP1, the product of the brown locus, is indeed a tyrosinase with DHICA oxidase activity. However, as opposed to the tyrosinase encoded by the albino locus, TRP1's role in melanogenesis could be more directly related to DHICA metabolism than to the first steps of the pathway.
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PMID:A new enzymatic function in the melanogenic pathway. The 5,6-dihydroxyindole-2-carboxylic acid oxidase activity of tyrosinase-related protein-1 (TRP1). 802 58

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight "multimers" of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.
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PMID:High-molecular-weight forms of tyrosinase and the tyrosinase-related proteins: evidence for a melanogenic complex. 804 Jun 9

Two groups of S-[2-(N,N-dialkylamino)ethyl]isothiourea derivates which depigmented melanoma cells either with inhibition of tyrosinase (group 1, R = methyl, isopropyl) or without inhibition of tyrosinase (group 2, R = benzyl, phenyl) were studied. Treatment of human melanoma cells with non-lethal doses of group 1 drugs led to a reduction in the levels of mRNA for the pigmentation genes tyrosinase, tyrosinase-related protein-1 and Pmel 17. The group 1 drug S-[2-N,N-diisopropylamino)ethyl[isothiourea] (DINOR) (R = isopropyl) produced only moderate inhibition of DNA, RNA and protein synthesis in three cell lines during the first 24 hr of treatment, and there was no correlation between the extent of inhibition and long-term toxicity. A group 2 drug (R = benzyl) rapidly inhibited DNA synthesis in an amelanotic melanoma cell line (MM96E) sensitive to killing by the drug; association of the latter with inhibition of RNA or protein synthesis was less clear. MM96E cells were also sensitive to killing by reactive oxygen species. In pigmented melanoma cells (MM418), incorporation of [125I]thiouracil, a false precursor of melanin, increased during the first 24 hr of treatment with DINOR whereas a group 2 drug (R = phenyl) inhibited incorporation of [125I]thiouracil. Cells depigmented by treatment with drugs from either group suffered the same amount of DNA damage as pigmented cells after UVB irradiation, as judged by inhibition of DNA synthesis, but did not recover as well as pigmented cells, whether or not drug was present during recovery. The results suggested that (1) group 1 agents down-regulated message for several pigmentation genes, possibly at the transcriptional level; (2) the toxicity of group 2 drugs was related to reactive oxygen species; and (3) melanin protected cells from UVB by enhancing cellular recovery.
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PMID:Reduction of DNA synthesis, pigment synthesis, pigmentation gene mRNA and resistance to UVB in human melanoma cells treated with analogues of a histamine (H2) agonist. 804 13

Retinoic acid (RA) is a hormone-like agent involved in the control of cell differentiation. The most characteristic feature of melanocyte differentiation, melanogenesis, is stimulated by UV radiations. Excessive chronic sun exposure results in irregular skin hypermelanosis that can be partially corrected by topical RA. The basic mechanisms underlying this effect of RA are unknown. To determine whether RA can directly modulate excessive melanin synthesis, we analyzed the in vitro effect of cis- and trans-RA on UVB-induced melanogenesis in S91 mouse melanoma cells and in normal human melanocytes (NHM). In both cells types, the two RA isoforms significantly decreased the UVB-stimulated melanogenesis in term of tyrosinase activity and melanin neosynthesis. To correlate changes in melanogenesis with the expression of melanogenic enzymes, we determined the neosynthesis rate of tyrosinase, tyrosinase-related protein-1 (TRP-1/gp 75) and tyrosinase-related protein-2 (TRP-2/DOPAchrome tautomerase). Here we show that UVB-induced melanogenesis in NHM is related to an increased synthesis of tyrosinase and TRP-1 and to a dramatic decrease of TRP-2 expression. RA inhibition of UVB-induced melanogenesis acts at the post-transcriptional level leading to a decreased tyrosinase and TRP-1 synthesis. We also show that in NHM, inhibition of TRP-2 following UVB-treatment is significantly reversed by RA. This demonstrates a negative correlation between melanogenesis and TRP-2 expression.
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PMID:Retinoic acid as modulator of UVB-induced melanocyte differentiation. Involvement of the melanogenic enzymes expression. 805 33

Human melanocytes from individuals with different skin types, as well as from the skin of the same individual, are heterogeneous in their melanin content. This heterogeneity may be attributed to differences in the activity and expression of the three melanogenic proteins: tyrosinase and tyrosinase-related proteins 1 and 2 (gp75 and DOPAchrome tautomerase, respectively), which in turn are affected by certain regulatory factors. Established melanocyte strains that exhibited intrinsic melanogenic heterogeneity could be separated into subpopulations according to density and melanin content by Percoll density gradient centrifugation. The least melanotic subpopulation consisted of melanocytes that contained an active tyrosinase enzyme and a low amount of melanin. Tyrosinase activity and the quantities of tyrosinase enzyme, tyrosinase-related protein-1 and DOPAchrome tautomerase gradually increased with increased melanin content and Percoll density of the isolated melanocyte subpopulations. We have found a direct correlation between melanin content, tyrosinase activity and the expression of the three melanogenic proteins in melanocyte strains established from different skin types. Addition of the two epidermal cytokines, tumor necrosis factor-alpha or interleukin-1 alpha, to cultures of human melanocytes from different skin types caused decreased proliferation, tyrosinase activity and expression of tyrosinase, tyrosinase-related protein-1 and DOPAchrome tautomerase. Similar results were obtained when Percoll-derived melanocyte subpopulations were treated with tumor necrosis factor-alpha and interleukin-1 alpha. These results indicate that the variation in melanin content in human melanocytes is due to differences in the activity and expression of the melanogenic proteins, which are influenced by autocrine and paracrine factors.
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PMID:Contribution of melanogenic proteins to the heterogeneous pigmentation of human melanocytes. 812 11

Using melanotic cells (SK-MEL-23 and G361) and amelanotic cells (C32 and SK-MEL-24) of human melanoma, this study examined whether UV-B irradiation has a direct stimulatory effect on the expression of genes involved in melanogenesis. Our initial screening of methylthiazol tetrazolium (MTT)-formazan formation assay indicated a low dose of ultraviolet (UV)-B irradiation, 2.5 and 5.0 mJ/cm2, can metabolically stimulate these cells. Repeated exposure of UV-B at 5.0 mJ/cm2 for seven consecutive days resulted in increased tyrosinase activity and melanin synthesis in SK-MEL-23 and G361 cells, but not in C32 and SK-MEL-24 cells. On reverse-transcription-polymerase chain reaction and immunoprecipitation studies, the two melanotic cell lines exhibited upregulated expression of mRNA and antigenic epitopes of tyrosinase, tyrosinase-related protein (TRP-1; gp75/HMSA-5), and lysosomal membrane associated protein (Lamp-1). The amelanotic cell line, C32, expressed the tyrosinase gene and protein constitutively but revealed no active tyrosinase or melanin synthesis even after UV-B exposure. Another amelanotic cell line, SK-MEL-24, exhibited no expression of tyrosinase gene and protein before and after UV-B exposure and, therefore, no melanin synthesis. Both C32 and SK-MEL-24 showed no gene or protein expression of TRP-1 before or after UV exposure, but upregulation of the Lamp-1 gene and protein expressions after exposure. We conclude that tyrosinase is the key enzyme responsible for UVB-induced melanogenesis. Both TRP-1 and Lamp-1 act together in melanogenesis, TRP-1 being essential and necessary. There is no change in the expression of CD63 lysosomal membrane protein at either the mRNA or protein level.
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PMID:Role of gene expression and protein synthesis of tyrosinase, TRP-1, lamp-1, and CD63 in UVB-induced melanogenesis in human melanomas. 815 Nov 27

Antiserum raised in rabbits against the Triton X-100 insoluble fraction of melanosomes from mouse melanoma cells specifically decorates the internal matrix of melanosomes in immunoelectron microscopy. In metabolic labeling studies, the antiserum recognizes a protein of 94 kDa, which is processed to a band of 53 kDa. Whereas the precursor is relatively soluble in buffers containing Triton X-100, the processed protein requires the addition of sodium dodecyl sulfate for effective solubilization, as would be expected for a melanosomal matrix constituent. Tunicamycin reduces the Mr of the nascent protein to 75 kDa, but deoxymannojirimycin and swainsonine have no effect, suggesting that following initial glycosylation in the endoplasmic reticulum, the protein is not subject to processing by glycosidases in the Golgi apparatus or may bypass it entirely. Subcellular fractionation followed by immunoblotting confirms that the protein is present in the melanosome-rich, large granule fraction. Expression of the protein is regulated differently from that of the tyrosinase-related protein family. Conditions that greatly stimulate expression of tyrosinase-related proteins do not affect matrix protein expression, nor is the protein immunologically related to the tyrosinase-related protein family. Our results suggest that we have identified an authentic component of the mammalian melanosomal matrix, and that its characteristics lend support to a bipartite pathway for melanosomal biogenesis.
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PMID:Identification of a mammalian melanosomal matrix glycoprotein. 834 14


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