Comparative Analysis of Melanins and Melanosomes Produced by Various Coat Color Mutants

Pigmentation in the skin, hair, and eyes of animals is influenced by a number of genes that modulate the activity of melanocytes, the intervention of enzymatic controls at different stages of the melanogenic process, and the physico-chemical properties of the final pigment. The results of combined phenotypic, ultrastructural, biochemical, and chemical analyses of hairs of a variety of defined genotypes on a common genetic background performed in this study are consistent with the view that pigmentation of dark to black hairs results from the incorporation of eumelanin pigments whereas that of yellow hairs results from the incorporation of eu- and pheomelanins. It is also clear that relatively minor differences in melanin content can have dramatic effects on visible hair color. A good correlation was found for expression of (and enzyme activities associated with) TRP1 and TRP2 with eumelanin synthesis and eumelanosome production.     

植物病原真菌1,8-间苯二酚黑色素研究进展

黑色素是一种广泛分布于生物体中的酚类聚合物疏水色素,分为1,8-间苯二酚(1,8-dihydroxynaphthalene,DHN)黑色素和3,4-二羟基苯丙氨酸(3,4-dihydroxyphenylalanine,L-DOPA)黑色素两种,其中DHN黑色素多存在于子囊菌门的植物病原真菌中。基因组和转录组技术的发展及功能基因组研究的深入,使DHN黑色素合成途径上关键基因在不同病原真菌中被鉴定,而且黑色素与真菌抗逆、发育和致病的关系受到越来越多的关注。本文阐述了DHN黑色素合成途径及其在真菌抗辐射与抗极端温度中的作用,以及黑色素对真菌侵染和细胞发育的影响,旨在加深人们对黑色素介导真菌与环境和寄主协同进化的认识,这对黑色素的基础研究和开发利用具有重要意义。     

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.     

Evolution of albinism in cave planthoppers by a convergent defect in the first step of melanin biosynthesis

Albinism, the reduction or loss of melanin pigment, is found in many diverse cave‐dwelling animals. The mechanisms responsible for loss of melanin pigment are poorly understood. In this study we use a melanogenic substrate assay to determine the position where melanin synthesis is blocked in independently evolved cave planthoppers from Hawaii and Croatia. In this assay, substrates of enzymes responsible for melanin biosynthesis are added to fixed specimens in vitro and their ability to rescue black melanin pigmentation is determined. L‐tyrosine, the first substrate in the pathway, did not produce melanin pigment, whereas L‐DOPA, the second substrate, restored black pigment. Substrates in combination with enzyme inhibitors were used to test the possibility of additional downstream defects in the pathway. The results showed that downstream reactions leading from L‐DOPA and dopamine to DOPA‐melanin and dopamine‐melanin, the two types of insect melanin, are functional. It is concluded that albinism is caused by a defect in the first step of the melanin synthesis pathway in cave‐adapted planthoppers from widely separated parts of the world. However, Western blots indicated that tyrosine hydroxylase (TH), the only enzyme shown to operate at the first step in insects, is present in Hawaiian cave planthoppers. Thus, an unknown factor(s) operating at this step may be important in the evolution of planthopper albinism. In the cavefish Astyanax mexicanus, a genetic defect has also been described at the first step of melanin synthesis suggesting convergent evolution of albinism in both cave‐adapted insects and teleosts.     

The retinal pigment epithelium and photoreceptor cells light-and electron microscopic studies on monkey eyes

Summary In the perifoveal retina of the monkey, Cercopithecus aethiops, the melanin granules are accumulated in apical cytoplasmatic protrusions of the pigment epithelial cells, facing the end of the cones. The rods are inserted deeper into the pigment epithelium than the cones; they reach the bottom of the infoldings of the apical surface membrane of the pigment epithelial cells. No melanin granules or other inclusions are situated at the end of the rods. The outer extremity of the rods is considerably inclined and in sections often appears as groups of rod discs which are incompletely or completely separated from the main part of the outer segments. This separation is regarded as an artifact caused by the inclination of the rods, and it is therefore not considered to represent phagocytosis of the outer segments by the pigment epithelium.The inclusions of the pigment epithelial cells are classified in five categories which seem to be related to each other owing to their shared structural characteristics. It is suggested that melanin granules are produced, modified and destroyed by the pigment epithelial cells of the adult.Because of the relations between the photoreceptors and the melanin granules it is suggested that light scattered by the melanin granules may pass backwards through the outer segments of the cones, but not of the rods.This investigation was supported in part by the Danish Foundation for the Advancement of Science and by the Danish Medical Research Council.     

Electron Microscope Observations of the Melanocyte of the Human Epidermis

Using standard osmium fixation and methacrylate embedding techniques, a study has been made of the melanocyte of human biopsy skin removed under general and local anaesthesia. Melanogenesis was easily observable in the melanocytes, but immature pigment granules were rarely seen in the Malpighian cells. The passage of melanin from melanocyte to Malpighian cell—cytocrine secretion—is thought to have been observed. Phagocytes near the dermal-epidermal junction seem to have their pigment granules in vacuoles, rather than surrounded directly by the cytoplasmic matrix as in the melanocytes. This, together with the failure to observe "effete" melanocytes, prompts the suggestion that the phagocytes are melanocytes which have migrated from the epidermis into the dermis. A melanin granule is shown with alternating dark and lighter transverse striations, concerning which structure little can at present be said.     

Buthionine sulfoximine diverts the melanogenesis pathway toward the production of more soluble and degradable pigments

Buthionine sulfoximine (BSO) is a specific inhibitor of γ-glutamylcysteine synthetase, thus blocking the synthesis of glutathione (GSH). It is known that this makes that BSO affects melanin synthesis because of the role of thiols in melanogenesis. However, BSO may also react with the intermediate oxidation products of melanogenesis, a possibility that has not been investigated from the initial steps of the pathway. We created in vitro conditions simulating eumelanogenesis (oxidation of l-DOPA in the absence of GSH) and pheomelanogenesis (oxidation of l-DOPA in the presence of GSH) under presence or absence of BSO. BSO made that eumelanogenesis results in pigments more soluble and less resistant to degradation by hydrogen peroxide than pigments obtained without BSO. A similar but less marked effect was observed for pheomelanogenesis only at subsaturating concentrations of GSH. These results suggest that BSO diverts the melanogenesis pathway toward the production of more soluble and degradable pigments.     

Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones alpha-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation.     

The IFPCS presidential lecture: a chemist's view of melanogenesis

The significance of our understanding of the chemistry of melanin and melanogenesis is reviewed. Melanogenesis begins with the production of dopaquinone, a highly reactive o-quinone. Pulse radiolysis is a powerful tool to study the fates of such highly reactive melanin precursors. Based on pulse radiolysis data reported by Land et al. (J Photochem Photobiol B: Biol 2001;64:123) and our biochemical studies, a pathway for mixed melanogenesis is proposed. Melanogenesis proceeds in three distinctive steps. The initial step is the production of cysteinyldopas by the rapid addition of cysteine to dopaquinone, which continues as long as cysteine is present (1 microM). The second step is the oxidation of cysteinyldopas to give pheomelanin, which continues as long as cysteinyldopas are present (10 microM). The last step is the production of eumelanin, which begins only after most cysteinyldopas are depleted. It thus appears that eumelanin is deposited on the preformed pheomelanin and that the ratio of eu- to pheomelanin is determined by the tyrosinase activity and cysteine concentration. In eumelanogenesis, dopachrome is a rather stable molecule and spontaneously decomposes to give mostly 5,6-dihydroxyindole. Dopachrome tautomerase (Dct) catalyses the tautomerization of dopachrome to give mostly 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Our study confirmed that the role of Dct is to increase the ratio of DHICA in eumelanin and to increase the production of eumelanin. In addition, the cytotoxicity of o-quinone melanin precursors was found to correlate with binding to proteins through the cysteine residues. Finally, it is still unknown how the availability of cysteine is controlled within the melanosome.     

Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice

Albinism, due to a lack of melanin pigment, is one of the oldest known mutations in mice. Tyrosinase (monophenol oxygenase, EC 1.14.18.1) is the first enzyme in the pathway for melanin synthesis, and the gene encoding this enzyme has been mapped to the mouse albino (c) locus. We have used mouse tyrosinase cDNA clones and genomic sequencing to study the albino mutation in laboratory mice. Within the tyrosinase gene coding sequences, a G to C transversion at nucleotide 308, causing a cysteine to serine mutation at amino acid 103, is sufficient to abrogate pigment production in transgenic mice. This same base pair change is fully conserved in classical albino strains of laboratory mice. These results indicate that a conserved mutation in the tyrosinase coding sequences is responsible for the classical albino mutation in laboratory mice, and also that most albino laboratory mouse strains have been derived from a common ancestor.     

Regulation of Melanogenesis in Melanocytes

Melanogenesis refers to the biosynthesis of melanin pigment in pigment cells called melanocytes. Melanins are mixed biopolymers formed during a series of oxidation/reduction reactions that are initiated by the enzymatic hydroxylation of L-tyrosine to L-dopa. In living cells, melanogenesis is limited to melanosomes, the membrane bounded microscopic secretory granules of melanocytes. Melanosomes may be secreted into the environment as, for example, from the squid's ink gland; or be transferred to neighboring cells, such as the keratinocytes in human skin and hair; or they may remain within the pigment cell and change only their subcellular localization, as in the rapidly color-changing dermis of lower vertebrates. Regulation of the melanocytic phenotype involves synthesis of the biosynthetically active subcellular apparatus of melanogenesis, premelanosomes and tyrosinase, and the utilization of the final product, melanized melanosomes, in the translocation and secretory processes mentioned above. Genetic information for this regulation is stored in the nuclear genome whose expression is controlled by the intra- and extracellular environment. As premelanosomes become biosynthetically active, they mature into melanosomes by fusing with vesicles derived from the trans-Golgi network and the plasmalemma, thereby internalizing and incorporating contents and membrane components from inside the cell and the cell surface. In the process, melanosomes become acidified. The thesis pursued in this review explores the importance of the melanosome as the final common pathway of regulation of melanin biosynthesis.     

Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors

Melanogenesis is a process to synthesize melanin, which is a primary responsible for the pigmentation of human skin, eye and hair. Although numerous enzymatic catalyzed and chemical reactions are involved in melanogenesis process, the enzymes such as tyrosinase and tyrosinase-related protein-1 (TRP-1) and TRP-2 played a major role in melanin synthesis. Specifically, tyrosinase is a key enzyme, which catalyzes a rate-limiting step of the melanin synthesis, and the downregulation of tyrosinase is the most prominent approach for the development of melanogenesis inhibitors. Therefore, numerous inhibitors that target tyrosinase have been developed in recent years. The review focuses on the recent discovery of tyrosinase inhibitors that are directly involved in the inhibition of tyrosinase catalytic activity and functionality from all sources, including laboratory synthetic methods, natural products, virtual screening and structure-based molecular docking studies.     

The Interaction of Agouti Signal Protein and Melanocyte Stimulating Hormone to Regulate Melanin Formation in Mammals

Important regulatory controls of melanogenesis that operate at the subcellular level to modulate the structural and/or the functional nature of the melanins and melanin granules produced in melanocytes are reviewed. Melanocyte stimulating hormone and agouti signal protein have antagonistic roles and possibly opposing mechanisms of action in the melanocyte. In the mouse, melanocyte stimulating hormone promotes melanogenic enzyme function and elicits increases in the amount of eumelanins produced, while agouti signal protein reduces total melanin production and elicits the synthesis of pheomelanin rather than eumelanin. We are now beginning to understand the complex controls involved in regulating this switch at the molecular and biochemical levels. The quality and quantity of melanins produced by melanocytes have important physiological consequences for melanocyte function and undoubtedly play important roles in the various functions of the melanins per se, including hair and skin coloration and photoprotection.     

Modulating skin colour: role of the thioredoxin and glutathione systems in regulating melanogenesis

Different skin colour among individuals is determined by the varying amount and types of melanin pigment. Melanin is produced in melanocytes, a type of dendritic cell located in the basal layer of the epidermis, through the process of melanogenesis. Melanogenesis consists of a series of biochemical and enzymatic reactions catalysed by tyrosinase and other tyrosinase-related proteins, leading to the formation of two types of melanin, eumelanin and pheomelanin. Melanogenesis can be regulated intrinsically by several signalling pathways, including the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), stem cell factor (SCF)/c-kit and wingless-related integration site (Wnt)/β-catenin signalling pathways. Ultraviolet radiation (UVR) is the major extrinsic factor in the regulation of melanogenesis, through the generation of reactive oxygen species (ROS). Antioxidants or antioxidant systems, with the ability to scavenge ROS, may decrease melanogenesis. This review focuses on the two main cellular antioxidant systems, the thioredoxin (Trx) and glutathione (GSH) systems, and discusses their roles in melanogenesis. In the Trx system, high levels/activities of thioredoxin reductase (TrxR) are correlated with melanin formation. The GSH system is linked with regulating pheomelanin formation. Exogenous addition of GSH has been shown to act as a depigmenting agent, suggesting that other antioxidants may also have the potential to act as depigmenting agents for the treatment of human hyperpigmentation disorders.     

A Chemist's View of Melanogenesis

The significance of our understanding of the chemistry of melanin and melanogenesis is reviewed. Melanogenesis begins with the production of dopaquinone, a highly reactive o‐quinone. Pulse radiolysis is a powerful tool to study the fates of such highly reactive melanin precursors. Based on pulse radiolysis data reported by Land et al. (J Photochem Photobiol B: Biol 2001;64:123) and our biochemical studies, a pathway for mixed melanogenesis is proposed. Melanogenesis proceeds in three distinctive steps. The initial step is the production of cysteinyldopas by the rapid addition of cysteine to dopaquinone, which continues as long as cysteine is present (1 μM). The second step is the oxidation of cysteinyldopas to give pheomelanin, which continues as long as cysteinyldopas are present (10 μM). The last step is the production of eumelanin, which begins only after most cysteinyldopas are depleted. It thus appears that eumelanin is deposited on the preformed pheomelanin and that the ratio of eu‐ to pheomelanin is determined by the tyrosinase activity and cysteine concentration. In eumelanogenesis, dopachrome is a rather stable molecule and spontaneously decomposes to give mostly 5,6‐dihydroxyindole. Dopachrome tautomerase (Dct) catalyses the tautomerization of dopachrome to give mostly 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA). Our study confirmed that the role of Dct is to increase the ratio of DHICA in eumelanin and to increase the production of eumelanin. In addition, the cytotoxicity of o‐quinone melanin precursors was found to correlate with binding to proteins through the cysteine residues. Finally, it is still unknown how the availability of cysteine is controlled within the melanosome.     

Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ_m) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH₄Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state.     

Increase of melanogenesis by retinoic acid: an ultrastructural and morphometric study

Melanin is a dark pigment protecting the skin against UV radiation in some organisms. Studies on invasion and metastasis using retinoic acid as inhibitor agent are well known, but its role in melanin production (melanogenesis), especially at ultrastructural level and using morphometry were not well studied. In the present study, we analyzed the effects of retinoic acid on the melanosomes in B16F10 melanoma cells. These organelles were identified and quantified using routine electron microscopy and the specific HMB45 antibody. Other approaches such as immunofluorescence, and flow cytometry were also used. Our results indicated that retinoic acid increased the melanogenesis process in B16F10 melanoma cells. Furthermore, this work also provided evidence that this substance interferes at the subcellular level altering the numerical density of melanosomes, as well as the relative volume of the nucleus and nucleolus. In addition, the cells displayed altered morphology and an increase in the percentage of the relative volume of melanosomes, mainly the stages II-III and IV, leading to melanin formation. Furthermore, a decrease in the cells number after retinoic acid treatment was also observed.