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)

Specific CD8(+) CTL recognition of melanoma requires expression of MHC class I molecules as well as melanoma-associated peptide epitopes. Human melanoma cells may escape immune recognition by a variety of means, including global or allelic down-regulation of MHC class I molecules. Stable MHC class I cell surface expression requires delivery of cytosolic peptides into the endoplasmic reticulum by the peptide transporter molecules TAP1 and TAP2, with peptides subsequently transported to the cell surface in complexes containing MHC class I heavy chain and beta2-microglobulin. We have evaluated a series of mechanisms resulting in MHC class I down-regulation in a human melanoma cell line, Mz18, typed as HLA-A2(+), A3(+), B7(+), B57(+), Cw1(+), and Cw6(+) by genomic PCR analysis. The melanoma cell line Mz18 exhibits a global down-regulation of MHC class I heavy chain transcripts; beta2-microglobulin; the proteasome subunits LMP2/7, involved in generating cytosolic peptide fragments; and the peptide transporter molecules TAP1 and TAP2, involved in peptide transport from the cytosol into the endoplasmic reticulum. IFN-gamma treatment of Mz18 melanoma cells leads to up-regulation of LMP2/7 and TAP1/2, as well as to up-regulation of HLA-B and HLA-C MHC loci alleles, but not HLA-A2 or HLA-A3. Karyotypic analysis and fluorescence in situ hybridization with chromosome 6 and MHC class I-specific probes showed complex rearrangement of one chromosome 6 involving the MHC class I locus on 6p and translocation of 6q to the long arm of chromosome 19. To evaluate the capability of melanoma Mz18 to present tumor-specific peptides to HLA-A2-restricted, melanoma-specific CTLs, we restored HLA-A2 surface expression by retroviral-mediated transfer of functional HLA-A2 cDNA. Melanoma peptides could only be presented and recognized by CTLs if the HLA-A2-transfected Mz18 cell line was first treated with IFN-gamma, thereby restoring LMP2/7 and TAP1/2 expression and function. Because several melanoma antigens recognized by T cells have been reported to be presented by HLA-A2 (MART-1/Melan-A, tyrosinase, gp100, and MAGE-3), the loss of HLA-A2 molecules may represent an important mechanism by which many melanomas evade immune recognition. These findings suggest that patients entering clinical trials for immunotherapy with melanoma vaccines should be carefully examined for tumor cell allelic MHC class I loss and whether such MHC class I antigen down-regulation can be restored by cytokines.
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PMID:Tumor escape from immune recognition: loss of HLA-A2 melanoma cell surface expression is associated with a complex rearrangement of the short arm of chromosome 6. 981 14

To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.
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PMID:Promotion of tyrosinase folding in COS 7 cells by calnexin. 988 Aug 1

Tyrosinase and tyrosinase-related protein-1 (TRP-1) are two melanogenic enzymes that regulate melanin biosynthesis. Both are glycoproteins and belong to the TRP-1 gene family. They share a significant level of sequence similarity in several regions, including the catalytic domain and the potential N-glycosylation sites. We have recently shown that inhibition of the early steps of N-glycan processing in B16F1 cells dramatically affects tyrosinase activity and melanin synthesis. We present here results on N-glycan processing of TRP-1 and tyrosinase and compare the maturation process and activity of both glycoproteins in the presence of inhibitors of the endoplasmic reticulum stages of N-glycosylation. N-glycan analysis reveals that each of these two glycoproteins contains a mixture of high-mannose and sialylated complex N-glycans. However, in contrast to TRP-1, tyrosinase presents a homogeneous high-mannose glycoform, also. In the presence of alpha-glucosidases inhibitors, the maturation of tyrosinase N-glycans is completely inhibited, whereas TRP-1 is still able to acquire some complex glycans, indicating that endomannosidase acts preferentially on the later glycoprotein. In addition, the dopa-oxidase activity of tyrosinase is totally abolished, whereas for TRP-1 it is only partially affected. The results suggest that despite their structural similarity, tyrosinase is more sensitive than TRP-1 to perturbations of early N-glycan processing, in terms of maturation and catalytical activity.
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PMID:Protein specific N-glycosylation of tyrosinase and tyrosinase-related protein-1 in B16 mouse melanoma cells. 1058 52

Antigenic peptides have been found associated with heat shock proteins (HSP) including cytoplasmic HSP70 and heat shock cognate protein 70 as well as the endoplasmic reticulum-resident glucose-regulated protein 94. Recently, HSP70 transfection has been reported to increase MHC class I cell surface expression and antigen presentation on mouse melanoma B16 cells (Wells et al., Int. Immunol. 1998. 10: 609). To analyze the effect of HSP70 on MHC class I cell surface expression and lysability of target cells we transfected a human melanoma cell line with the rat Hsp70-1 gene using the Tet-On system for conditional overexpression of HSP70. Induction of HSP70 did not increase cell surface expression of HLA class I molecules in general or individual HLA-A and B antigens in particular. Nonetheless, induction of HSP70 enhanced susceptibility of these cells to lysis by allospecific CTL. The same effect was observed using an HLA-A2-restricted tyrosinase-specific CTL clone after pulsing the tyrosinase-negative target cells with the specific peptide. Thus, HSP70 induction can increase killing by CTL without affecting MHC class I cell surface expression or antigen processing. This effect of HSP70 appears to be different from the commonly found protection exerted by HSP70 against stress like heat shock, and might be mediated by improving CTL-induced apoptosis.
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PMID:Enhanced susceptibility to cytotoxic T lymphocytes without increase of MHC class I antigen expression after conditional overexpression of heat shock protein 70 in target cells. 1060

Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37 degrees C but displays full activity in cells grown at 31 degrees C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37 degrees C but exits the ER and accumulates in endosomal structures in cells grown at 31 degrees C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37 degrees C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37 degrees C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.
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PMID:A common temperature-sensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature. 1076 67

Tyrosinase is a melanocyte-specific enzyme critical for the synthesis of melanin, a process normally restricted to a post-Golgi compartment termed the melanosome. Loss-of-function mutations in tyrosinase are the cause of oculocutaneous albinism, demonstrating the importance of the enzyme in pigmentation. In the present study, we explored the possibility that trafficking of albino tyrosinase from the endoplasmic reticulum (ER) to the Golgi apparatus and beyond is disrupted. Toward this end, we analyzed the common albino mouse mutation Tyr(C85S), the frequent human albino substitution TYR(T373K), and the temperature-sensitive tyrosinase TYR(R402Q)/Tyr(H402A) found in humans and mice, respectively. Intracellular localization was monitored in albino melanocytes carrying the native mutation, as well as in melanocytes ectopically expressing green fluorescent protein-tagged tyrosinase. Enzymatic characterization of complex glycans and immunofluorescence colocalization with organelle-specific resident proteins established that all four mutations produced defective proteins that were retained in the ER. TYR(R402Q)/Tyr(H402A) Golgi processing and transport to melanosomes were promoted at the permissive temperature of 32 degrees C, but not at the nonpermissive 37 degrees C temperature. Furthermore, evidence of protein misfolding was demonstrated by the prolonged association of tyrosinase mutants with calnexin and calreticulin, known ER chaperones that play a key role in the quality-control processes of the secretory pathway. From these results we concluded that albinism, at least in part, is an ER retention disease.
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PMID:Endoplasmic reticulum retention is a common defect associated with tyrosinase-negative albinism. 1082 41

Newly synthesized melanosomal proteins, like many other cellular proteins, traverse through a series of intracellular compartments en route to melanosomes. Entry and exit of proteins through these compartments is orchestrated by cellular sorting machinery that recognize specific sorting signals. Melanosomal membrane proteins begin their intracellular journey upon co-translational importation into the endoplasmic reticulum (ER). The biosynthetic output of tyrosinase, the key melanogenic enzyme, appears to be regulated by quality-control events at the ER, the 'port of entry' to the secretory pathway. Following maturation in the ER and through the Golgi, the sorting of these proteins in the trans-Golgi network for intracellular retention and transport along endosome/lysosome pathway requires cytoplasmically exposed signals. A di-leucine motif, present in the cytoplasmic tails of most melanosomal proteins, and its interaction with adaptor protein (AP) complexes, specifically AP-3, are critical for these events. Defects in sorting signals and the cytosolic components that interact with these signals result in a number of murine coat color phenotypes and cause human pigmentary disorders. Thus, missense or frame-shift mutations that produce truncated tyrosinase lacking the melanosomal sorting signal(s) appear to be responsible for murine platinum coat color phenotypes and a proportion of human oculocutaneous albinism-1; mutations in AP-3 appear to be responsible for the mocha phenotype in mice and Hermansky-Pudlak-like syndrome in man. Additional signals and sorting steps downstream of AP-3 appear to be required for endosomal sorting and targeting proteins to melanosomes. Signals and mechanisms that sequester melanosomal proteins from endosomes/lysosomes are not understood. Potential candidates that mediate such processes include proteins encoded by lyst and pallid genes. The common occurrence of abnormalities in melanosomes in many storage-pool disorders suggests that melanocytes utilize signals, pathways, and mechanisms shared by other proteins and cell types to assemble a number of specialized proteins and produce unique cell-type-specific organelles.
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PMID:Sorting and targeting of melanosomal membrane proteins: signals, pathways, and mechanisms. 1088 69

In this study we have explored the endoplasmic reticulum associated events accompanying the maturation of the tyrosinase-related protein-1 (TRP-1) nascent chain synthesized in mouse melanoma cells. We show that TRP-1 folding process occurs much more rapidly than for tyrosinase, a highly homologous protein, being completed post-translationally by the formation of critical disulfide bonds. In cells pretreated with dithiothreitol (DTT), unfolded TRP-1 is retained in the endoplasmic reticulum by a prolonged interaction with calnexin and BiP before being targeted for degradation. The TRP-1 chain was able to fold into DTT-resistant conformations both in the presence or absence of alpha-glucosidase inhibitors, but folding occurred through different pathways. During the normal folding pathway, TRP-1 interacts with calnexin. In the presence of alpha-glucosidase inhibitors, the interaction with calnexin is prevented, with TRP-1 folding being assisted by BiP. In this case, the process has similar kinetics to that of untreated TRP-1 and yields a compact form insensitive to DTT as well. However, this form has different thermal denaturation properties than the native conformation. We conclude that disulfide bridge burring is crucial for the TRP-1 export. This suggests that although various folding pathways may complete this process, the native form may be acquired only through the normal unperturbed pathway.
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PMID:Folding and maturation of tyrosinase-related protein-1 are regulated by the post-translational formation of disulfide bonds and by N-glycan processing. 1091 99

The intracellular vesicular trafficking in the melanosome biogenesis (melanogenesis) is reviewed with the incorporation of our own experimental findings. The melanosome biogenesis involves four stages of melanosome maturation, which reflect the transport of structural and enzymatic proteins from Golgi (trans-Golgi network: TGN) to the melanosomal compartment and their organization therein. The major melanosomal proteins include tyrosinase gene family protein (tyrosinase and tyrosinase-related protein; TRP), lysosome-associated membrane protein (Lamp) and gp100 (pmel 17). They are glycosylated in the endoplasmic reticulum, and transported by vesicles from the TGN to the melanosomal compartment. During the formation of transport vesicles, they assemble on the cytoplasmic face of the TGN to select cargo by interacting directly or indirectly with coat proteins. Tyrosinase and TRP-1 possess the dileucine motifs at the cytoplasmic domain, to which adapter protein-3 binds to transport them from the TGN to stage I melanosomes (related to late endosomes) and then to stage II melanosomes. A number of small guanosine triphosphate-binding proteins, including rab 7, appear to be involved in this vesicular transport. Phosphatidyl inositol 3 kinase also regulates this membrane trafficking of melanosomal glycoprotein. Eumelanogenesis is controlled by melanocyte-stimulating hormone, and all three tyrosinase gene family proteins are transported from the TGN to stage II melanosomes that are elliposoidal and contain the structural matrix of filaments/lamellae. In contrast, pheomelanogenesis is primarily regulated by agouti signal protein, and only tyrosinase is transported from stage I melanosomes to stage II melanosomes that are spherical and related to lysosomes. Because of the absence of TRP-1 and TRP-2 in pheomelanogenesis, it may be suggested that tyrosinase is involved in lysosomal degradation after forming dopaquinone, to which the cysteine present in the lysosomal granule binds to form cysteinyldopas that will then be auto-oxidized to become pheomelanin.
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PMID:Intracellular vesicular trafficking of tyrosinase gene family protein in eu- and pheomelanosome biogenesis. 1104 67

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or seven N-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential cotranslational maturation events.
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PMID:Translation rate of human tyrosinase determines its N-linked glycosylation level. 1106 24


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