title: Investigation of the intracellular transport of tyrosinase and tyrosinase related protein (TRP)-1. The effect of endoplasmic reticulum (ER)-glucosidases inhibition.
abstract: Melanin biosynthesis is completely inhibited in the B16 melanoma cells following their incubation with inhibitors of the two ER glucosidases. This is primarily due to the inactivation of tyrosinase. Under the same conditions, the DOPA-oxidase activity of TRP-1 was only partially affected. In this report we investigate the effects of the perturbation of N-glycan processing in ER on the transport and activation of tyrosinase and TRP-1. We have localized the DOPA-oxidase activity in normal and inhibited cells and suggest that the first DOPA-reactive compartment of the secretory pathway (trans Golgi network) is also the site of tyrosinase activation. The inhibition of N-glycan processing does not affect the intracellular trafficking of the two melanogenic enzymes that are correctly transported to melanosomes. Immunoprecipitation experiments followed by analysis in SDS-PAGE under non-reducing conditions suggest that in inhibited cells, both tyrosinase and TRP-1 are synthesized in a modified conformation as compared to the normal proteins. These data suggest that the inhibition of melanin synthesis is not due to a defective transport but rather to conformational changes induced in the structure of tyrosinase and TRP-1 during their transit through the ER.
authors.affiliation: Institute of Biology, Splaiul Independentei 296, Bucharest, Romania.
authors.forename: G
authors.initials: G
authors.lastname: Negroiu
authors.forename: N
authors.initials: N
authors.lastname: Branza-Nichita
authors.forename: G E
authors.initials: GE
authors.lastname: Costin
authors.forename: H
authors.initials: H
authors.lastname: Titu
authors.forename: A J
authors.initials: AJ
authors.lastname: Petrescu
authors.forename: R A
authors.initials: RA
authors.lastname: Dwek
authors.forename: S M
authors.initials: SM
authors.lastname: Petrescu
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005959:Glucosidases / Q000037:antagonists & inhibitors / Q000502:physiology*; D006031:Glycosylation / Q000187:drug effects; D007424:Intracellular Fluid / Q000378:metabolism; D008546:Melanoma, Experimental / Q000378:metabolism / Q000473:pathology; D020460:Melanosomes / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D008854:Microscopy, Electron; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011506:Proteins; D017485:1-Deoxynojirimycin; C059896:miglustat; D010088:Oxidoreductases; C079621:Tyrp1 protein, mouse; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D005959:Glucosidases