Melanosomal Proteins – Role in Melanin Polymerization

Melanin, the major determinant of skin colour, is a tyrosine‐based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post‐tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin–protein interaction is not nonspecific.     

Melanosomal proteins--role in melanin polymerization

Melanin, the major determinant of skin colour, is a tyrosine-based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post-tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin-protein interaction is not nonspecific.     

Interactions of flavins with melanin. Studies on equilibrium binding of riboflavin to dopa-melanin and some spectroscopic characteristics of flavin-melanin complex

Natural melanins are photoprotective pigments that in mammals are principally found in the skin, hair, and eyes. Although the molecular mechanism of photoprotection of pigmented cells has not yet been established, several hypotheses have been proposed with melanin acting as a light filter, free radical scavenger, and quencher of electronically excited states of reactive intermediates. It can be expected that the detoxicating efficiency of melanin should be enhanced if the melanin and potentially cytotoxic species are brought close together. Such a situation may occur for a number of photosensitizing dyes that have the ability to bind to melanin. The interaction of melanin with flavins has been studied under strictly controlled experimental conditions. The equilibrium dialysis method has been employed to determine dissociation constants and the number of binding sites in melanin at pH 5-9. The data reveal that synthetic DOPA-melanin has two different classes of binding sites with dissociation constants of 10(-6) and 10(-5) M, respectively. The overall binding capacity of melanin, at pH 7, is 250 nmol RF/mg melanin. The amount of bound-to-melanin RF increases with pH. The absorption spectra of melanin complexes with RF and lumiflavin indicate that hydrophobic interaction may be involved in the binding of these flavins by melanin. No changes in flavin fluorescence have been detected after binding of flavin to melanin. It appears that, contrary to cationic photosensitizing dyes, the singlet excited state of flavin molecules is not quenched by melanin.     

Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns

BACKGROUND: Animal melanin patterns are involved in diverse aspects of their ecology, from thermoregulation to mimicry. Many theoretical models have simulated pigment patterning, but little is known about the developmental mechanisms of color pattern formation. In Drosophila melanogaster, several genes are known to be necessary for cuticular melanization, but the involvement of these genes in melanin pattern evolution is unknown. We have taken a genetic approach to elucidate the developmental mechanisms underlying melanin pattern formation in various drosophilids. RESULTS: We show that, in D. melanogaster, tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are required for melanin synthesis. Ectopic expression of TH, but not DDC, alone was sufficient to cause ectopic melanin patterns in the wing. Thus, changes in the level of expression of a single gene can result in a new level of melanization. The ontogeny of this ectopic melanization resembled that found in Drosophila species bearing wing melanin patterns and in D. melanogaster ebony mutants. Importantly, we discovered that in D. melanogaster and three other Drosophila species these wing melanin patterns are dependent upon and shaped by the circulation patterns of hemolymph in the wing veins. CONCLUSIONS: Complex wing melanin patterns are determined by two distinct developmental mechanisms. Spatial prepatterns of enzymatic activity are established late in wing development. Then, in newly eclosed adults, melanin precursors gradually diffuse out from wing veins and are oxidized into dark brown or black melanin. Both the prepatterning and hemolymph-supplied components of this system can change during evolution to produce color pattern diversity.     

Role of light in human skin color viariation.

The major source of color in human skin derives from the presence within the epidermis of specialized melanin-bearing organelles, the melanosomes. Tanning of human skin on exposure to ultraviolet light results from increased amounts of melanin within the epidermis. Melanosomes synthesized by melanocytes are acquired by keratinocytes and transported within them to the epidermal surface. In some cases, the melanosomes are catobolized en route. New information indicates that the multicellular epidermal melanin unit (melanocyte and associated pool of keratinocytes) rather than the melanocyte alone is the focal point for the control of melanin metabolism within mammalian epidermis. Gross human skin color derives from the visual impact of the summed melanin pigmentation of the many epidermal melanin units. In theory, constitutive skin color in man designates the genetically-determined levels of melanin pigmentation developed in the absence of exposure to solar radiation or other environmental influences; facultative skin color or "tan" characterizes the increases in melanin pigmentation above the constitutive level induced by ultraviolet light. The details of genetic regulation of pigment metabolism within the epidermal melanin units are being clarified. In some mammals at least, the function of epidermal melanin units is significantly influenced by hormones which may be regulated by radiations received through the eyes. Based on an evolutionary history of the human family which exceeds ten million years, it is proposed that melanin pigmentation may have played a number of roles in human adaptions to changing biologic and physical environments.     

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.     

Melanin standard method: empirical formula.

Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.     

Melanin inhibits cytotoxic effects of doxorubicin and daunorubicin in MOLT 4 cells

In the present study we have developed a simple method to elucidate the melanin binding ability of different chemotherapeutic agents. The anthracyclines, doxorubicin and daunorubicin, or the alkylating agent cisplatin were preincubated with melanin (Sepia). Melanin and free drug was then separated through centrifugation and the cytotoxic effects of corresponding drug were evaluated in a MTT (3-(4,5-dimetyltiazol-2-yl)-2,5-difenyl-tetrazoliumbromide) assay using MOLT-4 cells. Our results show that melanin pretreatment shifted the IC50 value for doxorubicin from 0.06 to 0.97 microM and for daunorubicin from 0.04 to 0.80 microM. In contrast, the IC50 values of cisplatin was not influenced by melanin pre-treatment indicating that cisplatin does not bind to melanin. By comparing equi-active concentrations from concentration-response curves with or without melanin pretreatment an approximate binding capacity of melanin could be estimated. Our results show that melanin binds about 900 nmol/mg doxorubicin and 760 nmol/mg daunorubicin. Chloroquine, which is known to bind to melanin with high affinity, was found to inhibit melanin binding of both daunorubicin and doxorubicin, thereby leading to an increased sensitivity of the anthracyclines. The clinical implications of melanin binding regarding unwanted accumulation of anthracyclines in the skin as well as chemoprotective effects against chemotherapy are discussed.     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.     

Revisiting the condition‐dependence of melanin‐based plumage

Support against the condition‐dependence of melanin plumage signals has relied on data from species exhibiting both melanin‐ and carotenoid‐based plumage ornaments. As the mechanisms leading to variation in carotenoid‐ and melanin‐based plumage differ fundamentally, these systems may not be ideal to assess the condition‐dependence of melanin signals. Instead, we hypothesized that melanin‐plumage is more likely to signal condition in purely achromatic species. We performed a meta‐analysis reviewing evidence for condition‐dependent melanic plumage: we compared the net effect size for the relationship between melanin traits and condition in species that are achromatic versus species that also display a carotenoid‐based trait. Our results indicate that melanin plumage is condition‐dependent in species of both plumage types. Contrarily to our prediction, this finding suggests that melanin ornament condition‐dependence is not conditional on the context of other ornaments within a species. Instead, melanin ornaments should be viewed as potential condition‐dependent signals in all species.     

Inhibition of transglutaminase by synthetic tyrosine melanin

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. Previously, we found a high molecular mass transglutaminase-inhibitory substance produced by Streptomyces lavendulae Y-200 that appeared to be a melanin substance. Here, we report that synthetic tyrosine melanin inhibited various types of transglutaminases. Tyrosine melanin inhibited tissue-type transglutaminase in a competitive manner with a glutamine substrate, and also inhibited the cross-linking of casein catalyzed by a tissue-type transglutaminase. The melanized hemolymph of the silkworm and melanin solutions prepared from melanin precursors inhibited tissue-type transglutaminase. These results suggested that the melanin substances generally inhibit transglutaminases.     

Decolorization of synthetic melanins by crude laccases of Lentinus polychrous L��v.

Melanins are complex natural pigments that darken the skin and are difficult to degrade. This study evaluated synthetic melanin decolorization by the crude laccase from fungus Lentinus polychrous in the absence and presence of selected redox mediators. The greatest melanin decolorization activity was 87?% at pH?6.5 within 3?h in the presence of 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS), whereas only about 22?% melanin decolorized at pH?5.0 in case of no mediator. The optimum temperatures for melanin decolorization in the absence and presence of ABTS were 55 and 35°C, respectively. Using a natural redox mediator, 1.0?mmol/L vanillin leads to 45?% melanin decolorization. Our results suggest the possibility of applying vanillin for L. polychrous laccase-catalyzed decolorization of melanin.     

Melanin Standard Method: Empirical Formula

Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.     

PDE5 inhibitor promotes melanin synthesis through the PKG pathway in B16 melanoma cells

PDE inhibitors could increase cellular cGMP levels and are used to treat erectile dysfunction as well as pulmonary arterial hypertension. cGMP production was reported to be necessary for UVB-induced melanin synthesis, however, the effect of PDE5 inhibitor on melanin synthesis has not been examined. We found that PDE5 inhibitor (sildenafil or vardenafil) and the cGMP analog 8-CPT-cGMP stimulated CREB phosphorylation, leading to increased tyrosinase expression and melanin synthesis, which was counteracted by KT5823, a selective cGMP-dependent protein kinase (PKG) inhibitor. However, KT5823 did not affect cAMP-elevating agent-mediated melanin synthesis, indicating that KT5823 selectively inhibited cGMP-induced melanin synthesis. This is the first study to find that PDE5 inhibitor can promote melanin synthesis and reveal that PKG-dependent CREB phosphorylation and tyrosinase expression is involved in cGMP-induced melanin synthesis. Our results suggest that PDE5 inhibitor may be beneficial for the treatment of hypopigmentation diseases.     

Does Melanin Turnover Occur in the Eyes of Adult Vertebrates?

This paper is a review of what is known about the turnover of melanin in iris, choroid, and retinal pigment epithelium (RPE) of the adult vertebrate eye. Differences in size and structure of choroideal and retinal pigment epithelial melanin granules are shown by electron micrographs. The classical stages of melanin synthesis, including the premelanosome, are shown in the RPE of adult hamsters that had been exposed to intense light. Degradation or synthesis of melanin also seem to occur in the melanocytes of the choroid in these animals. It is postulated that all three pigmented eye tissues (iris, RPE, and choroid) of adult vertebrates form melanin granules in vivo. However, nothing is known about the amount of this turnover.     

Electron Spin Resonance Studies on Melanin

Electron spin resonance (e.s.r.) observations of squid melanin have been conducted over the temperature range 500°K to 4.2°K, and the effect of various chemical treatments of the melanin upon the e.s.r. spectrum has been studied. The findings have shown that the paramagnetism of this melanin follows the Curie Law from 500°K to 4.2°K, that the spin signal can be eliminated by the addition of Cu⁺⁺ to the melanin, and that the optical and e.s.r. absorptions of melanin are independent since either can be reduced or eliminated without affecting the other. Similar studies on synthetic melanins produced by autoxidation or by enzymatic oxidation of a number of biphenols were carried out. It was found that the e.s.r. signals of these synthetic melanins were strikingly similar (with respect to line width, line shape, and g-value) with those of squid melanin. It is concluded that the unpaired electrons observed are associated with trapped free radicals in the melanin polymer, that the biosynthesis of melanin may involve a free radical mechanism, and that these physical data are in accord with the concept of Nicolaus that melanin is a highly irregular, three-dimensional, polymer.     

Fluorescent quantification of melanin

Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non‐melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Therefore, fluorescence spectroscopy is the best method for melanin quantification as it proved to be highly specific and accurate, detecting even small variations in the synthesis of melanin. This method can also be applied to the quantification of melanin in more complex biological matrices like zebrafish embryos and human hair.     

Melanin pigments of fungi under extreme environmental conditions (Review)

This review is dedicated to the research on the functions of melanin pigments in fungi. The participation of melanin pigments in protection from environmental factors is considered. Data on the biosynthetic pathways and types of melanin pigments in fungi are presented.     

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of melanin synthesis and secretion by B16/C3 melanoma cells

In culture, B16/C3 murine melanoma cells grown in the presence of serum undergo melanogenesis at a specific time after plating. At this time, melanin is synthesized intracellularly and then secreted into the extracellular culture fluid. We have found that melanin secretion is dependent on the presence of serum in the growth medium. When confluent cultures are deprived of serum, that is, refed with serum-free medium, cells remain viable but do not undergo melanogenesis. Addition of serum-free medium supplemented with either melanocyte-stimulating hormone (MSH) or dibutyryl cAMP induced melanogenesis in these cells but did not result in melanin secretion. Furthermore, when B16/C3 cells are grown in serum-free, hormone-supplemented medium, they also undergo melanogenesis but fail to release melanin. The addition of serum, however, to B16/C3 cells induced to undergo melanogenesis with MSH, dibutyryl cAMP, or hormone-supplemented medium promotes melanin secretion. Fractionation studies hence revealed that serum contains specific factors capable of inducing melanin secretion. These results demonstrate that factors that regulate melanin synthesis are distinct from those that induce cells to release melanin into their extracellular environment. Furthermore, the ability to induce melanogenesis with single factors will permit us to study the precise sequence of events leading to differentiation in B16/C3 cells under chemically defined conditions.