José C. García-Borrón

Melanins and melanogenesis: methods, standards, protocols

Despite considerable advances in the past decade, melanin research still suffers from the lack of universally accepted and shared nomenclature, methodologies, and structural models. This paper stems from the joint efforts of chemists, biochemists, physicists, biologists, and physicians with recognized and consolidated expertise in the field of melanins and melanogenesis, who critically reviewed and experimentally revisited methods, standards, and protocols to provide for the first time a consensus set of recommended procedures to be adopted and shared by researchers involved in pigment cell research. The aim of the paper was to define an unprecedented frame of reference built on cutting‐edge knowledge and state‐of‐the‐art methodology, to enable reliable comparison of results among laboratories and new progress in the field based on standardized methods and shared information.

Molecular cloning and functional characterization of a unique multipotent polyphenol oxidase from Marinomonas mediterranea

Marinomonas mediterranea is a recently isolated melanogenic marine bacterium containing laccase and tyrosinase activities. These activities are due to the expression of two polyphenol oxidases (PPOs), a blue multicopper laccase and an SDS-activated tyrosinase. The gene encoding the first one, herein denominated M. mediterranea PpoA, has been isolated by transposon mutagenesis, cloned and expressed in Escherichia coli. Its predicted amino acid sequence shows the existence of a signal peptide and four copper-binding sites characteristic of the blue multicopper proteins, including all fungal laccases. In addition, two additional putative copper-binding sites near its N-terminus are also present. Recombinant expression in E. coli of this protein clearly demonstrates its multipotent capability, showing both laccase-like and tyrosinase-like activities. This is the first prokaryotic laccase sequenced and the first PPO showing such multipotent catalytic activity. The expression of several truncated products indicates that the four copper-binding sites typical of blue multicopper proteins are essential for the laccase activity of this enzyme. However, the last two of these sites are not necessary for tyrosine hydroxylase activity as this activity is retained in a truncated product containing the first two sites as well as the extra histidine-rich clusters close to the N-terminus of the protein.

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.

Melanins and melanogenesis: from pigment cells to human health and technological applications

During the past decade, melanins and melanogenesis have attracted growing interest for a broad range of biomedical and technological applications. The burst of polydopamine‐based multifunctional coatings in materials science is just one example, and the list may be expanded to include melanin thin films for organic electronics and bioelectronics, drug delivery systems, functional nanoparticles and biointerfaces, sunscreens, environmental remediation devices. Despite considerable advances, applied research on melanins and melanogenesis is still far from being mature. A closer intersectoral interaction between research centers is essential to raise the interests and increase the awareness of the biomedical, biomaterials science and hi‐tech sectors of the manifold opportunities offered by pigment cells and related metabolic pathways. Starting from a survey of biological roles and functions, the present review aims at providing an interdisciplinary perspective of melanin pigments and related pathway with a view to showing how it is possible to translate current knowledge about physical and chemical properties and control mechanisms into new bioinspired solutions for biomedical, dermocosmetic, and technological applications.

MC1R, the cAMP pathway, and the response to solar UV: extending the horizon beyond pigmentation

The melanocortin 1 receptor (MC1R) is a G protein‐coupled receptor crucial for the regulation of melanocyte proliferation and function. Upon binding melanocortins, MC1R activates several signaling cascades, notably the cAMP pathway leading to synthesis of photoprotective eumelanin. Polymorphisms in the MC1R gene are a major source of normal variation of human hair color and skin pigmentation, response to ultraviolet radiation (UVR), and skin cancer susceptibility. The identification of a surprisingly high number of MC1R natural variants strongly associated with pigmentary phenotypes and increased skin cancer risk has prompted research on the functional properties of the wild‐type receptor and frequent mutant alleles. We summarize current knowledge on MC1R structural and functional properties, as well as on its intracellular trafficking and signaling. We also review the current knowledge about the function of MC1R as a skin cancer, particularly melanoma, susceptibility gene and how it modulates the response of melanocytes to UVR.

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.

Thr40 and Met122 are new partial loss‐of‐function natural mutations of the human melanocortin 1 receptor

Activation by melanocortins of the melanocortin 1 receptor (MC1R), expressed in epidermal melanocytes, stimulates melanogenesis. Human MC1R gene loss‐of‐function mutations are associated with fair skin, poor tanning and increased skin cancer risk. We identified two natural alleles: Ile40Thr, probably associated with skin types I–II, and Val122Met. Val122Met bound [125I][Nle4, D‐Phe7]‐α‐melanocyte stimulating hormone with lower affinity than the wild‐type. Dose–response curves of cAMP accumulation were right‐shifted for both forms. The Val122Met form failed to achieve maximal cAMP responses comparable to the wild‐type or Ile40Thr receptors. Thus, the Ile40Thr and Val122Met variants are partial loss‐of‐function natural mutations of MC1R.

Identification and functional analysis of novel variants of the human melanocortin 1 receptor found in melanoma patients.

The melanocortin 1 receptor, a Gs protein‐coupled receptor expressed in epidermal melanocytes, is a major determinant of skin pigmentation and phototype and an important contributor to melanoma risk. MC1R activation stimulates synthesis of black, strongly photoprotective eumelanin pigments. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation, and increased skin cancer risk. The MC1R gene is highly polymorphic, but only a few naturally occurring alleles have been functionally characterized, which complicates the establishment of accurate correlations between the signaling properties of mutant alleles and defined cutaneous phenotypes. We report the functional characterization of six MC1R alleles found in Spanish melanoma patients. Two variants (c.152T>C, p.Val51Ala and c.865T>C, p.Cys289Arg) have never been described, and the others (c.112G>A, p.Val38Met; c.122C>T, p.Ser41Phe; c.383T>C, p.Met128Thr; and c.842A>G, p.Asn281Ser) have not been analyzed for function. p.Asn281Ser corresponds to a functionally silent polymorphism. The other mutations are associated with varying degrees of loss of function (LOF), from moderate decreases in coupling to the cAMP pathway (p.Val38Met and p.Val51Ala) to nearly complete absence of functional coupling (p.Ser41Phe, p.Met128Thr, and p.Cys289Arg). The LOF p.Met128Thr and p.Cys289Arg mutants are trafficked to the cell surface, but are unable to bind agonists efficiently. Conversely, LOF of p.Val38Met, p.Ser41Phe, and p.Val51Ala is due to reduced cell surface expression as a consequence of retention in the endoplasmic reticulum (ER). Therefore, LOF of MC1R alleles is frequently associated with aberrant forward trafficking and accumulation within the ER or with inability to bind properly the activatory ligand. Hum Mutat 30:1–12, 2009.

Regulation of Human Melanocortin 1 Receptor Signaling and Trafficking by Thr-308 and Ser-316 and Its Alteration in Variant Alleles Associated with Red Hair and Skin Cancer

The melanocortin 1 receptor (MC1R), a G protein-coupled receptor (GPCR) positively coupled to adenylyl cyclase, is a key regulator of melanocyte proliferation and differentiation and a determinant of pigmentation, skin phototype, and skin cancer risk. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to yellowish and poorly photoprotective pheomelanin pigments. Desensitization and internalization are key regulatory mechanisms of GPCR signaling. Agonist-induced desensitization usually depends on phosphorylation by a GPCR kinase (GRK) followed by receptor internalization in endocytic vesicles. We have shown that MC1R desensitization is mediated by two GRKs expressed in melanocytes and melanoma cells, GRK2 and GRK6. Here we show that in contrast with this dual specificity for desensitization, GRK6 but not GRK2 mediated MC1R internalization. Mutagenesis studies suggested that the targets of GRK6 are two residues located in the MC1R cytosolic C terminus, Thr-308 and Ser-316. A T308D/S316D mutant mimicking their phosphorylated state was constitutively desensitized and associated with endosomes, whereas a T308A/S316A mutant was resistant to desensitization and internalization. We studied the desensitization and internalization of three variant MC1R forms associated with red hair and increased skin cancer risk: R151C, R160W, and D294H. These variants showed a less efficient desensitization. Moreover, D294H was resistant to internalization, thus accounting for its abnormally high surface expression. Co-expression of variant and wild type MC1R modified its desensitization and internalization behavior. These data suggest that MC1R might be regulated by novel mechanisms including differential effects of GRKs and altered desensitization rates of certain allelic combinations.

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