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.     

The neuromelanin of human substantia nigra: physiological and pathogenic aspects

Neuromelanin (NM) accumulates as a function of age in normal human substantia nigra (SN) but is relatively depleted in the SN of patients with Parkinson disease (PD). Several studies have been performed to further our understanding of the role of NM in neuronal aging and neurodegenerative mechanisms of PD. To this purpose, NM from human SN was isolated and its structure and molecular interactions were investigated. Cysteinyl-dopamine was shown to be one precursor of NM synthesis. A striking affinity of NM for specific metals, lipids, drugs and pesticides was found in vitro, and in animal and human brain postmortem studies. Because of these affinities, NM seems to play a protective role in the human brain by blocking toxic molecules. On the other hand, experiments in cell culture indicate that NM can activate microglia, eliciting the release of cytotoxic factors that can induce neurodegeneration.     

The structure of neuromelanin as studied by chemical degradative methods

The biosynthesis, structure and function of neuromelanin (NM), the dark brown melanin-like pigment present in the substantia nigra (SN), are not well characterized, in spite of the possible involvement of NM in the etiology and pathogenesis of Parkinson's disease. NM was isolated from the SN of five non-Parkinsonian human brains. NM and synthetic melanins, employed as models, were characterized by chemical analysis. Alkaline hydrogen peroxide (H2O2) oxidation of NM generated four degradation products, pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), thiazole-4,5-dicarboxylic acid (TDCA) and thiazole-2,3,5-tricarboxylic acid (TTCA), whose ratios, especially the TTCA to PDCA ratio, indicate that NM is derived mostly from dopamine (DA) with 25% incorporation of cysteine (Cys) in the form of a benzothiazine structure. Hydriodic acid (HI) reductive hydrolysis of NM yielded 4-amino-3-hydroxyphenylethylamine (4-AHPEA) as a marker of cysteinyldopamine (CysDA)-derived units. The 4-AHPEA to PDCA ratio indicates a 21% incorporation of CysDA-derived units into NM. These degradative experiments also suggest that DOPA is not incorporated into NM to a significant extent (approximately 6% the level of DA). It is concluded that the TTCA to PDCA ratio is a useful indicator of CysDA-derived units in NM, and NM consists mainly of DA-melanin with some contribution from CysDA-melanin. The involvement of DA and CysDA as building blocks of NM demonstrates the detoxifying role of NM synthesis, since it prevents the intraneuronal accumulation of DA and CysDA, which would cause toxic effects.     

Changes in the proliferation and differentiation of neonatal mouse pink-eyed dilution melanocytes in the presence of excess tyrosine

Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild-type at the pink-eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum-free primary culture, with or without the addition of L-Tyr. Incubation with added L-Tyr inhibited the proliferation of P/P melanocytes in a concentration-dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L-Tyr stimulated the proliferation of p/p melanoblasts-melanocytes derived from 0.5-day-old mice, but inhibited their proliferation when derived from 3.5- or 7.5-day-old mice. L-Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5-, 3.5- and 7.5-day-old p/p mice, but L-Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole-2,3,5-tricarboxylic acid as well as the pheomelanin marker, 4-amino-3-hydroxyphenylalanine in p/p melanocytes was greatly reduced compared with P/P melanocytes. However, the contents of eumelanin and its precursor, 5,6-dihydroxyindole-2-carboxylic acid, as well as the contents of pheomelanin and its precursor, 5-S-cysteinyldopa in culture media from p/p melanocytes were similar to those of P/P melanocytes at all ages tested. L-Tyr increased the content of eumelanin and pheomelanin two- to threefold in cultured cells and media derived from 0.5-, 3.5- and 7.5-day-old mice. These results suggest that the proliferation of p/p melanoblasts-melanocytes is stimulated by L-Tyr, and that the differentiation of melanocytes is induced by L-Tyr as the age of the donor mice advanced, although eumelanin and pheomelanin fail to accumulate in p/p melanocytes and are released from them at all ages of skin development.     

Change in the concentration of vitamins C and E in rat tissues by paraquat administration

Paraquat causes lung injury by oxidative stress. After 48 h of intraperitoneal administration of paraquat (50 mg/kg of body weight) to rats, the vitamin C concentration in the lungs was significantly decreased, while lung vitamin E content was increased after 12 h. These results indicate that vitamin C directly reflected the oxidative stress in the lungs.     

Involvement of 101F6, a homologue of cytochrome b561, in the reduction of ferric ions

We have isolated and characterized a small transmembrane protein, called 101F6, showing high sequence homology to cytochrome b(561), a protein containing two binding sites for haem. The newly identified 101F6 contains six membrane spanning domains in which conserved histidine residues are located, and has a molecular mass of 25 kDa. When the haem-binding with bacterial expressed 101F6 was examined, the protein bound haem and the deletion of one histidine residue at 149 caused a loss of the binding. 101F6 mRNA was expressed widely in various tissues, and especially abundant in liver, kidney and lung. It was also expressed in several cultured cell lines. The protein expressed from the 101F6 cDNA in Balb/3T3 cells was about 25 kDa in size and was localized in small vesicles, including endosomes and endoplasmic reticulum of the perinuclear region. Comparison of the location of 101F6 with that of transferrin receptor-1 revealed that the localization of 101F6 in small vesicles was not always the same as the localization of transferrin receptor-1, but was similar to that of haem oxygenase-1. The other homologue to cytochrome b(561), SDR-2 was also expressed in the small vesicles similar to the location of 101F6. Finally, reduction of ferric ions as well as of azo-dye increased with 101F6- or SDR-2-expressing cells. Thus, both 101F6 and SDR-2 were localized in small vesicles of cells and played roles in the reduction of ferric ions.     

Mechanisms of DNA damage induced by morin,an inhibitor of amyloid β-peptide aggregation

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer’s disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on ³²P-5′-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical ?OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H₂O₂ generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.     

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene‐350 and high‐performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole‐type pheomelanin) as well but not 4‐AHP (degradation product of benzothiazine‐type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.     

Tyrosinase‐catalyzed metabolism of rhododendrol (RD) in B16 melanoma cells: production of RD‐pheomelanin and covalent binding with thiol proteins

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was reported to induce leukoderma of the skin. To explore the mechanism underlying that effect, we previously showed that oxidation of RD with mushroom tyrosinase produces RD‐quinone, which is converted to secondary quinone products, and we suggested that those quinones are cytotoxic because they bind to cellular proteins and produce reactive oxygen species. We then confirmed that human tyrosinase can oxidize both enantiomers of RD. In this study, we examined the metabolism of RD in B16F1 melanoma cells in vitro. Using 4‐amino‐3‐hydroxy‐n‐butylbenzene as a specific indicator, we detected moderate levels of RD‐pheomelanin in B16F1 cells exposed to 0.3 to 0.5 mM RD for 72 h. We also confirmed the covalent binding of RD‐quinone to non‐protein thiols and proteins through cysteinyl residues. The covalent binding of RD‐quinone to proteins was 20‐ to 30‐fold greater than dopaquinone. These results suggest that the tyrosinase‐induced metabolism of RD causes melanocyte toxicity.     

Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle

The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.