José A. Lozano

The 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase activity of human tyrosinase.

Melanin synthesis in mammals is catalysed by at least three enzymic proteins, tyrosinase (monophenol dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and tyrosinase-related proteins (tyrps) 1 and 2, whose genes map to the albino, brown and slaty loci in mice, respectively. Tyrosinase catalyses the rate-limiting generation of L-dopaquinone from L-tyrosine and is also able to oxidize L-dopa to L-dopaquinone. Conversely, mouse tyrp1, but not tyrosinase, catalyses the oxidation of the indolic intermediate 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the corresponding 5,6-indolequinone-2-carboxylic acid, thus promoting the incorporation of DHICA units into eumelanin. The catalytic activities of the human melanogenic enzymes are still debated. TYRP1 has been reported to lack DHICA oxidase activity, whereas tyrosinase appears to accelerate DHICA consumption, thus raising the question of DHICA metabolism in human melanocytes. Here we have used two different approaches, comparison of the catalytic activities of human melanocytic cell lines expressing the full set of melanogenic enzymes or deficient in TYRP1, and transient expression of TYR and tyr genes in COS7 cells, to demonstrate that human tyrosinase actually functions as a DHICA oxidase, as opposed to the mouse enzyme. Therefore, human tyrosinase displays a broader substrate specificity than its mouse counterpart, and might be at least partially responsible for the incorporation of DHICA units into human eumelanins.

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-β1 (TGF-β1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-β1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-β1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-β1 on the melanogenic activity of B16/F10 cells.

Comparative action of dopachrome tautomerase and metal ions on the rearrangement of dopachrome

A vis-a-vis comparison between the effects of dopachrome tautomerase (DCT) and metal ions, e.g., cupric ions, on the kinetics and mode of rearrangement of dopachrome has been carried out under appropriate analytical conditions. The enzyme-promoted reaction is highly stereospecific for L-dopachrome, is unaffected by metal chelators and has an optimal pH around 6.8. By contrast, the kinetics of dopachrome rearrangement catalysed by cupric ions are not dependent on the stereochemistry of the substrate, are affected by EDTA and are not influenced by the pH of the medium in the range between 5-7.5. Both cupric ions and DCT catalyse the rearrangement of dopachrome to give 5,6-dihydroxyindole-2-carboxylic acid (DICA) rather than 5,6-dihydroxyindole (DI). However, at comparable activity, the ratio of formation DICA/DI is significantly higher in the enzyme-catalysed than in the metal-catalysed reaction. These results provide an improved background to look into the mode of action of DCT and metal ions, enabling a clear cut differentiation between the effects of the two factors when both are present in biological extracts.

L-mimosine, a slow-binding inhibitor of mushroom tyrosinase

Abstract It was found that L -mimosine is a slow-binding inhibitor of L -DOPA oxidation by mushroom tyrosinase. This inhibition is characterized by a prolonged transient phase. A mechanism is postulated according to the kinetic data.

The mouse silver locus encodes a single transcript truncated by the silver mutation

In mammals, melanin synthesis occurs mainly in highly differentiated cells, the epidermal melanocytes, where it is restricted to specialized organelles called melanosomes. Among the melanosomal proteins known to participate in the control of melanogenesis, those encoded at the silver locus (Kwon et al. 1991) are obviously important, since their impairment by the silver (si) mutation in mice results in premature graying of the hair owing to loss of follicular melanocytes (Kwon et al. 1991), but their function is disputed. It has been proposed that they are structural matrix proteins of the melanosome (Kobayashi et al. 1994). Other roles have been claimed, either as “stablins”, proteins retarding melanogenesis by stabilization of biosynthetic intermediates, or as 5,6dihydroxyindole-2-carboxylic acid polymerases (Chakraborty et al. 1996). The homologous human SILV or PMEL17 gene (OMIM 155550, GDB 6277709) has been widely studied, since its protein products were proposed as potential markers of human melanoma and as immunotherapy targets. An apparently melanocyte-specific cDNA sequence was originally reported by Kwon et al. (1991; accession no. M77348, name PMEL17, also termed D12S53E). It was mapped to human Chromosome (Chr) 12pter-q21, sufficiently close to the murine silver locus (Chr 10; MGI 98301) to suggest similarity. The gene encodes a type 1 transmembrane protein of 668 amino acids, with a potential signal peptide, and putative membrane anchor domain near the C-terminus. Soon thereafter, other cDNA clones were reported encoding a melanocyte-specific protein almost identical to PMEL17, termed either gp100 (as a 100-kDa glycoprotein; Adema et al. 1994), or ME20M (Maresh et al. 1994; accession no. M32295). These products were identical to each other. The amino acid differences from PMEL17 were one substitution (P274L) and the deletion of a heptapeptide (VPGILLT) located just before the transmembrane region (amino acids 588–594). Partial sequence analysis of genomic DNA indicated that gp100 and PMEL17 transcripts originated from a single gene via alternative splicing (Adema et al. 1994). This was confirmed once the full genomic organization and sequence of the human SILV locus were described (Bailin et al. 1996; Kim et al. 1996). The presence of the VPGILLT heptapeptide in PMEL17 but not in gp100 results from alternative mRNA splicing to two competing 38 splice acceptor sites, whose function and regulation are unknown. Both proteins share all other identifiable domains, including the cytosolic ExxPLL motif proposed as a melanosomal targeting signal (Xu et al. 1997), as well as ten Ser, Pro, and Thr-rich tandem repeats in the melanosomal portion. A murine si-derived cDNA named Pmel17 was also first cloned by Kwon et al. (1995; accession no. U14133). This sequence is called “Pmel17m” here for clarity. Comparison with the human sequences showed 77% nucleotide identity with PMEL17, but three main differences: (i) The central Ser/Thr/Pro-rich region is shorter in the murine protein; (ii) the predicted mouse “Pmel17m” protein lacks the heptapeptide distinguishing human PMEL17 from gp100, and so is actually a gp100 homolog; and (iii) there is a region of about 30 amino acids of low similarity to both human proteins in the C-terminal cytosolic domain. Other cDNAs encoding murine homologs of gp100 have been reported and were called “gp100” (Schreurs et al. 1997; Zhai et al. 1997). These were identical and similar to Pmel17m except that the 38 region of low similarity to the human sequences was missing. This was because in this region the murine gp100 sequence aligns numerically with the human, whereas there are three relative single-nucleotide deletions in Pmel17m, producing a local frameshift in the predicted protein compared with the human protein (Fig. 1, panel A). In the present work we will refer to this second murine gp100 homolog as “gp87” for clarity and in reference to the protein’s lower molecular weight determined by SDS-PAGE followed by Western immunoblotting (Schreurs et al. 1997, and data not shown). The murine silver mutation was described as a single base insertion in this same region, causing a frameshift and extension of the protein by 12 amino acids (Kwon et al. 1995). The predicted mutant protein lacks the putative melanosomal targeting signal, suggesting misrouting of the protein. Interestingly, this insertion occurs exactly at the second position where Pmel17m has a deletion relative to gp87 (Fig. 1, panel A). The reported differences between gp87 and Pmel17m are surprising and difficult to explain. It has not been shown whether these sequences can be expressed simultaneously or are allelic, nor whether any murine homolog of PMEL17, the longer human splicing variant, exists. This lack of knowledge of the exact nature and relationships of the murine silver products is a serious drawback for certain immunotherapy studies. We have, therefore, reexamined both the normal and silver mutant sequences, at the cDNA and genomic levels. In order to detect murine silver transcripts, we first amplified cDNAs from B16-F10 melanoma cells, wild-type at the silver locus. The primers used (SF1/SR1) were common to the reported Pmel17m and gp87 transcripts and were chosen to amplify the Correspondence to: J.C. Garcia-Borron

Regulation of mammalian melanogenesis. II: The role of metal cations.

Melanogenesis can be divided into two phases. The first one involves two tyrosinase-catalyzed oxidations from tyrosine to dopaquinone and a very fast chemical step leading to dopachrome. The second phase, from dopachrome to melanin, can proceed spontaneously through several incompletely known reactions. However, some metal transition ions and protein factors different from tyrosinase might regulate the reaction rate and determine the structure and relative concentrations of the intermediates. The study of the effects of some divalent metal ions (Zn, Cu, Ni and Co) on some steps of the melanogenesis pathway has been approached using different radiolabeled substrates. Zn(II) inhibited tyrosine hydroxylation whereas Ni(II) and Co(II) were activators. Ni(II), Cu(II) and Co(II) accelerated chemical reactions from dopachrome but inhibited its decarboxylation. Dopachrome tautomerase also decreased decarboxylation. When metal ions and this enzyme act together, the inhibition of decarboxylation was greater than that produced by each agent separately, but amount of carboxylated units incorporated to the melanin was not higher than the amount incorporated in the presence of only cations. The amount of total melanin formed from tyrosine was increased by the presence of both agents. The action of Zn(II) was different from other ions also in the second phase of melanogenesis, and its effect on decarboxylation was less pronounced. Since tyrosine hydroxylation is the rate-limiting step in melanogenesis, Zn(II) inhibited the pathway. This ion seems to be the most abundant cation in mammalian melanocytes. Therefore, under physiological conditions, the regulatory role of metal ions and dopachrome tautomerase does not seem to be mutually exclusive, but rather complementary.

Dopachrome tautomerase decreases the binding of indolic melanogenesis intermediates to proteins

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.

Kinetic study in the transient phase of the suicide inactivation of frog epidermis tyrosinase.

This paper deals with the kinetic study of a multisubstrate mechanism with enzyme inactivation induced by a suicide substrate. A transient phase approach has been developed that enables the deduction of explicit equations of product concentration vs. time. From these equations kinetic constants which characterize the suicide substrate can be obtained. This study with tyrosinase enzyme, which acts on L-dopa and catechol allowed us to determine the corresponding kinetic parameters, indicating that catechol is about 8-times more powerful as a suicide substrate than is L-dopa.

Kinetic Characterization of Dopamine as a Suicide Substrate of Tyrosinase

A kinetic study of the inactivation of frog epidermis tyrosinase by a suicide substrate dopamine hydrochloride is described. The kinetic parameters and constants which characterize this reaction have been determined and the effects of pH and the stoichiometric inhibition by chloride have been considered.

Effect of detergents and endogenous lipids on the activity and properties of tyrosinase and its related proteins.

Within mammalian melanocytes, melanin biosynthesis is controlled by three enzymes structurally related: tyrosinase and two tyrosinase related proteins, TRP1 and TRP2. These melanosomal enzymes are integral membrane proteins with a carboxyl tail oriented to the cytoplasm, a single membrane-spanning helix and the bulk of the protein located inside the melanosome. Their solubilization is usually carried out by treatment of melanosomal preparations with non-ionic detergents, but, so far, no comparative study of the effect of the detergents employed on the properties of the solubilized proteins has been reported. We have compared the effect of the detergents Brij-35, Nonidet P-40, Tween-20, sodium deoxycholate and Triton X-114 on several properties of the melanogenic enzymes, including the solubilization yield, stability, electrophoretic behaviour and accessibility of epitopes located in the carboxyl tail to specific antibodies. Our data indicate that not only the total amount of enzymes solubilized, but also their relative proportions in the solubilized preparations depend on the detergent used. The non-ionic detergents apparently interact strongly with the melanogenic enzymes, affecting their mobility in SDS-PAGE, and might induce different conformations of the carboxyl tail. Complete replacement of lipids by the detergents results in a decreased stability that can be partially reversed by the addition of endogenous lipids. This treatment also produces a noticeable activation of the tyrosinase isoenzymes, which is higher for TRP1 than for tyrosinase. Taken together, these data show that the transmembrane and carboxyl fragments of the proteins of the tyrosinase family might modulate the stability and activity of the melanogenic enzymes.