RESUMO
BACKGROUND: Generation and accumulation of reactive oxygen/nitrogen species in the epidermis of patients with vitiligo has been widely documented. Moreover, semiquinone radical-mediated sensitivity has been shown in blood lymphocytes of these patients. OBJECTIVES: To determine the possible mechanism behind Q10-induced facial vitiligo. METHODS: This was a clinical assessment supported by in vivo Fourier transform-Raman spectroscopy and repigmentation. RESULTS: Topical Q10 application generated hydrogen peroxide (H2 O2 ) leading in turn to facial vitiligo in susceptible individuals. Proof of the basic result stemmed from reduction of epidermal H2 O2 by using narrowband ultraviolet B-activated propseudocatalase PC-KUS in association with cessation of depigmentation and repigmentation of the lost skin colour. CONCLUSIONS: Over-the-counter availability of Q10-containing topical formulations can be harmful to individuals susceptible to vitiligo.
Assuntos
Dermatoses Faciais/induzido quimicamente , Preparações Clareadoras de Pele/efeitos adversos , Ubiquinona/análogos & derivados , Vitiligo/induzido quimicamente , Administração Cutânea , Adulto , Catalase/administração & dosagem , Cosméticos/efeitos adversos , Feminino , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Pessoa de Meia-Idade , Medicamentos sem Prescrição/efeitos adversos , Preparações Clareadoras de Pele/administração & dosagem , Ubiquinona/administração & dosagem , Ubiquinona/efeitos adversos , Vitiligo/metabolismoRESUMO
Senile graying of human hair has been the subject of intense research since ancient times. Reactive oxygen species have been implicated in hair follicle melanocyte apoptosis and DNA damage. Here we show for the first time by FT-Raman spectroscopy in vivo that human gray/white scalp hair shafts accumulate hydrogen peroxide (H(2)O(2)) in millimolar concentrations. Moreover, we demonstrate almost absent catalase and methionine sulfoxide reductase A and B protein expression via immunofluorescence and Western blot in association with a functional loss of methionine sulfoxide (Met-S=O) repair in the entire gray hair follicle. Accordingly, Met-S=O formation of Met residues, including Met 374 in the active site of tyrosinase, the key enzyme in melanogenesis, limits enzyme functionality, as evidenced by FT-Raman spectroscopy, computer simulation, and enzyme kinetics, which leads to gradual loss of hair color. Notably, under in vitro conditions, Met oxidation can be prevented by L-methionine. In summary, our data feed the long-voiced, but insufficiently proven, concept of H(2)O(2)-induced oxidative damage in the entire human hair follicle, inclusive of the hair shaft, as a key element in senile hair graying, which does not exclusively affect follicle melanocytes. This new insight could open new strategies for intervention and reversal of the hair graying process.
Assuntos
Envelhecimento , Cor de Cabelo , Peróxido de Hidrogênio/metabolismo , Metionina/análogos & derivados , Estresse Oxidativo , Catalase/análise , Folículo Piloso/patologia , Humanos , Metionina/análise , Metionina/deficiência , Espécies Reativas de Oxigênio/metabolismo , RegeneraçãoRESUMO
Vitiligo occurs in Northern Europe in one of 200 people. The disease can cause significant psychological stress for the affected individual. These patients generate and accumulate massive amounts of H(2)O(2)- and peroxynitrite in the epidermal compartment. Consequently many proteins are oxidized or nitrated, leading in turn to partial or complete loss of functionality. Moreover, presence of DNA damage in the skin as well as in plasma has been shown, while apoptosis is not enhanced. Induction of DNA repair is associated with up-regulated functioning p53 protein. Considering possible genetic predisposition and /or spontaneous mutations, autoimmune reactions in the disease are put forward in the context of oxidative stress. In addition a review of recent and novel treatment modalities including the role of oxidative stress reduction and combined climatotherapy at the Dead Sea in a group are discussed.
Assuntos
Peróxido de Hidrogênio/imunologia , Modelos Imunológicos , Pele/imunologia , Proteína Supressora de Tumor p53/imunologia , Vitiligo/diagnóstico , Vitiligo/imunologia , Humanos , Vitiligo/terapiaRESUMO
Everyone knows and seems to agree that melanocytes are there to generate melanin - an intriguing, but underestimated multipurpose molecule that is capable of doing far more than providing pigment and UV protection to skin (1). What about the cell that generates melanin, then? Is this dendritic, neural crest-derived cell still serving useful (or even important) functions when no-one looks at the pigmentation of our skin and its appendages and when there is essentially no UV exposure? In other words, what do epidermal and hair follicle melanocytes do in their spare time - at night, under your bedcover? How much of the full portfolio of physiological melanocyte functions in mammalian skin has really been elucidated already? Does the presence or absence of melanocytes matter for normal epidermal and/or hair follicle functions (beyond pigmentation and UV protection), and for skin immune responses? Do melanocytes even deserve as much credit for UV protection as conventional wisdom attributes to them? In which interactions do these promiscuous cells engage with their immediate epithelial environment and who is controlling whom? What lessons might be distilled from looking at lower vertebrate melanophores and at extracutaneous melanocytes in the endeavour to reveal the 'secret identity' of melanocytes? The current Controversies feature explores these far too infrequently posed, biologically and clinically important questions. Complementing a companion viewpoint essay on malignant melanocytes (2), this critical re-examination of melanocyte biology provides a cornucopia of old, but under-appreciated concepts and novel ideas on the slowly emerging complexity of physiological melanocyte functions, and delineates important, thought-provoking questions that remain to be definitively answered by future research.
Assuntos
Melanócitos/fisiologia , Animais , Epiderme/fisiologia , Humanos , Queratinócitos/fisiologia , Melaninas/biossínteseRESUMO
The participation of (6R) 5,6,7,8-tetrahydrobiopterin (6-BH4) in regulating the tyrosine supply for melanin biosynthesis was investigated by the examination of human keratinocytes, melanocytes, and epidermal suction blisters from normal human skin and from patients with the depigmentation disorder vitiligo. Cells, as well as total epidermis, contained high phenylalanine hydroxylase activities and also displayed the capacity to synthesize and recycle 6-BH4, the essential cofactor for this enzyme. In vitiligo, 4a-hydroxy-BH4 dehydratase activity was extremely low or absent, yielding an accumulation of the nonenzymatic by-product 7-tetrahydrobiopterin (7-BH4) at concentrations up to 8 x 10(-6) M in the epidermis. This by-product is a potent competitive inhibitor in the phenylalanine hydroxylase reaction with an inhibition constant of 10(-6) M. Thus, 6-BH4 seems to control melanin biosynthesis in the human epidermis, whereas 7-BH4 may initiate depigmentation in patients with vitiligo.
Assuntos
Biopterinas/análogos & derivados , Epiderme/metabolismo , Melaninas/biossíntese , Vitiligo/metabolismo , Biopterinas/biossíntese , Biopterinas/metabolismo , Biopterinas/farmacologia , Diferenciação Celular , Células Cultivadas , GTP Cicloidrolase/metabolismo , Humanos , Queratinócitos/metabolismo , Melanócitos/metabolismo , Fenilalanina Hidroxilase/antagonistas & inibidores , Fenilalanina Hidroxilase/metabolismo , Tirosina/biossínteseRESUMO
Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.
Assuntos
Biopterinas/análogos & derivados , GTP Cicloidrolase/fisiologia , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Vitiligo/metabolismo , Biópsia , Biopterinas/biossíntese , Estudos de Casos e Controles , Catalase/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/fisiologia , Ativação Enzimática/efeitos dos fármacos , Epiderme/metabolismo , Epiderme/patologia , Retroalimentação Fisiológica/efeitos dos fármacos , GTP Cicloidrolase/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxirredução/efeitos dos fármacos , Vitiligo/patologiaRESUMO
The Ca(2+)-dependent precursor convertase furin is abundantly expressed in epidermal keratinocytes and melanocytes. In this context, it is noteworthy that proopiomelanocortin (POMC) cleavage is also processed by furin, leading to ACTH, beta-lipotropin, and beta-endorphin. All prohormone convertases including furin are regulated by Ca(2+). Because numerous epidermal peptides and enzymes are affected by H(2)O(2)-mediated oxidation, including the POMC-derived peptides alpha-MSH and beta-endorphin as shown in the epidermis of patients with vitiligo, we here asked the question of whether furin could also be a possible target for this oxidation mechanism by using immunofluorescence, RT-PCR, Western blotting, Ca(2+)-binding studies, and computer modeling. Our results demonstrate significantly decreased in situ immunoreactivity of furin in the epidermis of patients with progressive vitiligo (n = 10), suggesting H(2)O(2)-mediated oxidation. This was confirmed by (45)Ca(2+)-binding studies with human recombinant furin identifying the loss of one Ca(2+)-binding site from the enzyme after oxidation with H(2)O(2). Computer simulation supported alteration of one of the two Ca(2+)-binding sites on furin. Taken together, our results implicate that the Ca(2+)-dependent proteolytic activity of this convertase is targeted by H(2)O(2), which in turn could contribute to the reduced epidermal expression of the POMC-derived peptides alpha-MSH and beta-endorphin as documented earlier in patients with vitiligo.
Assuntos
Cálcio/metabolismo , Epiderme/metabolismo , Furina/metabolismo , Peróxido de Hidrogênio/farmacologia , Vitiligo/metabolismo , Sítios de Ligação , Células Cultivadas , Furina/química , Furina/genética , Humanos , Modelos Moleculares , Oxirredução , RNA Mensageiro/análiseRESUMO
The pathobiology of vitiligo has been hotly disputed for as long as one remembers, and has been a magnet for endless speculation. Evidently, the different schools of thought--ranging, e.g. from the concept that vitiligo essentially is a free-radical disorder to that of vitiligo being a primary autoimmune disease--imply very different consequences for the best therapeutic strategies that one should adopt. As a more effective therapy for this common, often disfiguring pigmentary disorder is direly needed, we must strive harder to settle the pathogenesis debate definitively--on the basis of sound experimental evidence, rather than by a war of dogmatic theories. Recognizing, however, that it is theories which tend to guide our experimental designs and choice of study parameters, the various pathogenesis theories on the market deserve to be critically, yet unemotionally re-evaluated. This Controversies feature invites you to do so, and to ask yourself: is there something important or worthwhile exploring in other pathogenesis scenarios than those already favoured by you that may help you improve your own study design, next time you have a fresh look at vitiligo? Vitiligo provides a superb model for the study of many fundamental problems in skin biology and pathology. Therefore, even if it later turns out that, as far as your own vitiligo pathogenesis concept is concerned, you have barked-up the wrong tree most of the time, chances are that you shall anyway have generated priceless new insights into skin function along the way.
Assuntos
Doenças Autoimunes/imunologia , Cálcio/metabolismo , Mutação/genética , Espécies Reativas de Oxigênio/metabolismo , Vitiligo/etiologia , Apoptose/fisiologia , Humanos , Melanócitos/imunologia , Melanócitos/metabolismo , Melanócitos/patologia , Estresse Oxidativo/fisiologia , Linfócitos T Citotóxicos/fisiologia , Vitiligo/genética , Vitiligo/metabolismoRESUMO
Human epidermal keratinocytes and melanocytes express proopiomelanocortins (POMC) and all of the enzymes for POMC processing, i.e. prohormone convertases PC-1 and PC-2 including the regulatory protein 7B2. In melanocytes POMC processing also occurs in the melanosome, a lysosome-derived organelle that specializes in the biosynthesis of melanin. Consequently, the autocrine synthesis and release of the key hormones ACTH, alpha and beta-MSH and beta-endorphin takes also place in melanocytes. All four hormones have been reported to promote the biosynthesis of eumelanin in melanocytes. ACTH and alpha-MSH bind to the melanocortin-1 receptor (MC-1-R) on the plasma membrane and activate the signalling pathway predominantly coupled to production of cAMP, and in some cell lines raising intracellular calcium levels. In the melanocyte this signalling is redundant due to the high expression of alpha1 and beta2-adrenoceptors. Downstream events increase melanocyte this signalling is redundant due to the high expression of a tyrosinase expression / activity to stimulate eumelanogenesis. Studies with rMC-1-R transfected COS cells showed that both ACTH and alpha-MSH bind to the receptor with similar or different affinity depending on the species (human vs mice). We have modelled the MC-1-R based on the X-ray crystal structure of a homologous 7 receptor rhodopsin. Docking studies with ACTH1-39, ACTH1-17 and ACTH11-17 and alpha-MSH1-13 revealed that all 3 ACTH peptides yield thermodynamically stable (key ACTH1-13 in-lock) complexes. Interestingly, alpha-MSH is predicted to only have a kinetic effect on the MC-1-R and beta-MSH has even a weaker affinity for the MC-1-R than alpha-MSH. Based on these results the relative importance of ACTH versus alpha-MSH in the human epidermis has been re-evaluated.
Assuntos
Melanócitos/metabolismo , Pró-Opiomelanocortina/metabolismo , Receptor Tipo 1 de Melanocortina/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Animais , Humanos , Hormônios Estimuladores de Melanócitos/metabolismo , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/metabolismo , Receptor Tipo 1 de Melanocortina/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Human tyrosinase (5.5 mg) has been purified from a single human melanotic melanoma metastasis (50.5 g). In the presence of dioxygen, L-tyrosine proved to be a very poor substrate for this enzyme with barely detectable activity compared to L-dopa. However, saturating superoxide anion (i.e., greater than 5 x 10(-3) M) enhanced the oxidation rate of L-tyrosine to dopachrome 40-fold. Hydrogen peroxide was shown to be a competitive inhibitor of tyrosinase when L-tyrosine was the substrate. This reversible inhibition is based on a slow pseudocatalase activity for tyrosinase. Monothiols and dithiols inhibit tyrosinase by different mechanisms. Reduced human thioredoxin and 2,3-dithiopropanol are allosteric inhibitors of tyrosinase yielding bis-cysteinate complexes with one of the copper atoms in the enzyme active site. Bis-cysteinate tyrosinase activity is down-regulated to 30% of native enzyme activity in the L-dopa assay; suggesting a true regulatory role for dithiols. Monothiols such as reduced glutathione and beta-mercaptoethanol are much less reactive with tyrosinase although 10(-3) M monothiol totally inhibits enzyme activity. Reduced thioredoxin inhibits tyrosinase 23-fold more than reduced glutathione under the same experimental conditions.
Assuntos
Peróxido de Hidrogênio/farmacologia , Indolquinonas , Monofenol Mono-Oxigenase/metabolismo , Compostos de Sulfidrila/farmacologia , Superóxidos/farmacologia , Ânions , Catalase/metabolismo , Cromatografia DEAE-Celulose , Humanos , Indóis/metabolismo , Levodopa/metabolismo , Melanoma/enzimologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/isolamento & purificação , Oxirredução , Quinonas/metabolismo , Especificidade por SubstratoRESUMO
Fotemustine is a novel chloroethylnitrosourea derivative currently used in Phase III clinical trials for disseminated metastatic melanoma. This drug has been shown to inhibit enzymes in the ribonucleotide reduction pathway (i.e., thioredoxin reductase, glutathione reductase and ribonucleotide reductase). 14C chloroethyl-labelled Fotemustine covalently labels the thiolate active sites of thioredoxin reductase and glutathione reductase yielding 14C chloroethyl-thioether enzyme-inhibitor complexes. Enzyme activities can be restored by a reduced thioredoxin or reduced glutathione mediated beta-elimination of the chloroethyl group. 14C Fotemustine has been used to determine its reactivity and metabolism in drug sensitive and resistant melanoma metastases and in cultures of sensitive and resistant clones of human melanoma cells. Melanoma metastases from four different patients who were treated with Fotemustine could be labelled with radioactive drug only under reducing conditions with NADPH as electron donor and DTNB as substrate. FPLC analysis of these extracts revealed two radioactive proteins (I) glutathione reductase and (II) an unidentified protein with 95 and 50 kDa subunits. A similar labelling pattern was also found in extracts of Fotemustine sensitive melanoma cells (Cal 1). Fotemustine resistant tumors were melanotic and contained more glutathione reductase than thioredoxin reductase, whereas sensitive tumors were clinically amelanotic with more thioredoxin reductase than glutathione reductase. Fotemustine resistant melanoma cells (Cal 7) showed a slower uptake of 14C-label with 34% less isotope intracellularly in 1 h compared to sensitive melanoma cells (Cal 1). These results strongly indicate (I) the induction of alternate electron donors thioredoxin reductase or glutathione reductase for ribonucleotide reduction determines tumor and melanoma cell responses to the drug and (II) Fotemustine transport and the intracellular redox status seems to regulate resistance in melanoma cells and tissues.
Assuntos
Antineoplásicos/farmacologia , Melanoma/metabolismo , Compostos de Nitrosoureia/farmacologia , Compostos Organofosforados/farmacologia , Antineoplásicos/metabolismo , Membrana Celular/enzimologia , Cromatografia Líquida de Alta Pressão , Resistência a Medicamentos , Glutationa Redutase/metabolismo , Humanos , Cinética , Melanoma/enzimologia , Melanoma/secundário , Compostos de Nitrosoureia/metabolismo , Compostos Organofosforados/metabolismo , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Electron spin resonance spectroscopy has been used to measure the reduction of nitroxide radicals on a spin-labeled quaternary ammonium substrate by plasma membrane-associated thioredoxin reductase (EC 1.6.4.5) at the surface of cutaneous and subcutaneous melanoma metastases from one patient (B.M.). Enzyme activity in these metastases was shown to be hyperactive compared to normal skin and was subject to inhibition by calcium. From the remainder of the tissue (50.6 g), plasma membrane-associated thioredoxin reductase has been isolated and its molecular properties were compared with the same enzyme purified from the cytosol of rat liver and Escherichia coli. The enzyme from melanoma possessed an identical molecular weight to that from rat liver as determined by SDS-polyacrylamide gel electrophoresis (Mr 58,000). Upon fluorescence spectroscopic examination, the enzyme from melanoma was shown to contain flavin adenine dinucleotide as previously shown in the enzymes from E. coli and rat liver. The increased activities in plasma membrane-associated thioredoxin reductase in metastases of malignant melanotic melanoma are discussed in terms of the cellular functions of this important enzyme.
Assuntos
Melanoma/enzimologia , NADH NADPH Oxirredutases/isolamento & purificação , Neoplasias Cutâneas/enzimologia , Tiorredoxina Dissulfeto Redutase/isolamento & purificação , Animais , Cálcio/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/enzimologia , Humanos , Melanoma/secundário , Membranas/enzimologia , Peso Molecular , RatosRESUMO
Thioredoxin reductase has been purified from human metastatic melanotic melanoma and amelanotic melanoma tissues. Enzyme from the melanotic melanoma tissue contains bound calcium showing classical sigmoidal allosteric kinetics, whereas enzyme from the amelanotic melanoma yielded normal Michaelis-Menten saturation with substrate. Calcium inhibition can be partially reversed by oxidized thioredoxin. 45Ca has been used to label the amelanotic melanoma enzyme in order to determine the number of calcium-binding sites. These isotope experiments yielded only one calcium-binding site per enzyme molecule. Enzyme labeled with 45Ca was dialyzed for 24 h without loss of radioactivity, but the addition of oxidized thioredoxin to this labeled enzyme caused 60% calcium exchange in 24 h. Comparative studies with Escherichia coli thioredoxin reductase showed similar calcium inhibition as well as partial reactivation with oxidized thioredoxin. The enzyme from E. coli previously sequenced by others, showed considerable homology with the first EF-hands calcium-binding site of calmodulin. Detailed calcium-binding studies indicated that 10(-5) M of this fast exchange ion was sufficient to cause allosteric regulation in 10 min. This strong calcium-binding property could explain the allosteric nature of the thioredoxin reductase purified from human metastatic melanotic melanoma and its role in the regulation of melanin biosynthesis.
Assuntos
Cálcio/metabolismo , Melanoma/enzimologia , NADH NADPH Oxirredutases/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Ácido Ditionitrobenzoico/farmacologia , Humanos , Cinética , Ligação Proteica , Espectrofotometria , Tiorredoxina Dissulfeto Redutase/isolamento & purificaçãoRESUMO
The nitrosoureas BCNU, CCNU, ACNU, and Fotemustine covalently deactivate thioredoxin reductase, glutathione reductase and ribonucleotide reductase by alkylating their thiolate active sites. Since thioredoxin reductase and glutathione reductase function as alternative electron donors in the biosynthesis of deoxyribonucleotides, catalyzed by ribonucleotide reductase, the inhibition of these electron transfer systems by the nitrosoureas could determine the cytostatic property of this homologous series of drugs. A detailed study of the kinetics and mechanism for the inhibition of purified thioredoxin reductases from human metastatic melanotic and amelanotic melanomas by the nitrosoureas showed significantly different inhibitor constants. This difference is due to the regulation of these proteins by calcium. Calcium protects thioredoxin reductase from deactivation by the nitrosoureas. In addition, it has been shown that reduced thioredoxin displaces the nitrosourea-inhibitor complex from the active site of thioredoxin reductase to fully reactivate enzyme purified from human metastatic amelanotic melanoma. It has been possible to label the active sites of thioredoxin reductase and glutathione reductase by using chloro[14C]ethyl Fotemustine, resulting in the alkylation of the thiolate active sites to produce chloro[14C]ethyl ether-enzyme inhibitor complexes. These complexes can be reactivated via reduced thioredoxin and reduced glutathione, respectively, by a beta-elimination reaction yielding [14C]ethylene and chloride ions as reaction products.
Assuntos
Antineoplásicos/farmacologia , Glutationa Redutase/antagonistas & inibidores , NADH NADPH Oxirredutases/antagonistas & inibidores , Compostos de Nitrosoureia/farmacologia , Oxirredutases , Ribonucleotídeo Redutases/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Transporte de Elétrons , Eritrócitos/enzimologia , Glutarredoxinas , Humanos , Cinética , Melanoma/enzimologia , Modelos Biológicos , Proteínas/metabolismo , Tiorredoxina Dissulfeto Redutase/isolamento & purificaçãoRESUMO
Thioredoxin reductase (TR) activity on primary melanomas and in surrounding skin is regulated by calcium and, therefore, TR activity can be used to measure the flux of calcium between primary tumors and their surrounding epidermis. Calcium uptake in human melanotic melanoma cell lines SKmel-23 (metastatic) and BC-PT-1 (primary) is related to the density of beta-2-adrenoceptors. The non-pigmented cell line HT-144 (metastatic), did not express beta-2-adrenoceptors, yielding a slow rate of calcium uptake compared to SKmel-23 and BC-PT-1. Cell extracts from melanotic and amelanotic melanoma tissues did not contain a phenylethanolamine-N-methyltransferase (PNMT) for the biosynthesis of epinephrine from norepinephrine and S-adenosylmethionine. However, human full-thickness skin, epidermis and cell cultures of human keratinocytes contained significant PNMT activities. Taken together, these results indicate that (a), TR can be used to monitor calcium flux between primary melanomas and their surrounding skin and vice versa and (b), calcium uptake may be regulated by stimulation of beta-2-adrenoceptors on melanotic melanomas by epinephrine synthesized in the surrounding skin.
Assuntos
Cálcio/metabolismo , Epinefrina/biossíntese , Melanoma/secundário , Neoplasias Cutâneas/metabolismo , Transporte Biológico , Biopterinas/análogos & derivados , Biopterinas/biossíntese , Membrana Celular/enzimologia , Células Cultivadas , Epinefrina/química , Humanos , Melanoma/metabolismo , Modelos Químicos , Feniletanolamina N-Metiltransferase/análise , Receptores Adrenérgicos beta/metabolismo , Tiorredoxina Dissulfeto Redutase/análise , Tiorredoxinas/metabolismoRESUMO
(6R)-L-erythro 5,6,7,8-tetrahydrobiopterin (6-BH4) and its 7-isomer (7-BH4) function as uncompetitive inhibitors of human and mushroom tyrosinases. Stoichiometry for the binding of [3H]-labeled 6-BH4 to both tyrosinases has been established as 1:1. Stable complexation of 6-BH4 to tyrosinase appears to involve a hydrophilic conserved glutamic acid (Glu131) with a pKa = 4.7. Photo-oxidation by UVB-light and O2 reverses the inhibition of tyrosinase by 6-BH4 and 7-BH4 with the 6-BH4/tyrosinase complex being four-fold more photolabile than 7-BH4/tyrosinase. The photo-oxidation of 6-BH4 by UVB-light can be assessed spectrophotometrically with this reaction yielding 7,8-dihydroxanthopterin as the final product, 7,8-Dihydroxanthopterin neither binds to nor inhibits tyrosinase. By contrast, UVA light does not catalyze the photodegradation of 6-BH4. Taken together, our results indicate that the photo-oxidation of the tetrahydrobiopterins by UVB may represent a photo-switch in the regulation of tyrosinase activity to promote de novo melanogenesis.
Assuntos
Biopterinas/análogos & derivados , Melaninas/biossíntese , Monofenol Mono-Oxigenase/metabolismo , Basidiomycota/enzimologia , Sítios de Ligação/fisiologia , Biopterinas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Fotólise , Ligação Proteica , Raios Ultravioleta , Xantopterina/análogos & derivados , Xantopterina/biossíntese , Xantopterina/metabolismoRESUMO
Patients with the depigmentation disorder vitiligo lack the capacity to synthesize the melanins from L-tyrosine via the essential activity of tyrosinase. The aim of this study has been to examine both the supply of the substrate (L-tyrosine) and the regulation of tyrosinase in the epidermis of subjects with vitiligo. Patients with this depigmentation disorder have a 3- to 5-fold increase in GTP-cyclohydrolase I activity leading to an excessive de novo synthesis of (6R)5,6,7,8 tetrahydrobiopterin (6-BH4). Continuous production of 6-BH-4 leads to: (1) an accumulation of the non-enzymatic byproduct 7-tetrahydropterin (7-BH4) in the epidermis, and (2) increased synthesis of the catecholamines in keratinocytes, leading to an excess of norepinephrine in both the plasma and urine of these patients. In vitiligo, the time-dependent production of 7-BH4 is caused by decreased 4a-hydroxytetrahydrobiopterin dehydratase activity; the essential enzyme for recycling and maintaining normal levels of 6-BH-4. In the epidermis and in cultured melanocytes, 7-BH4 is a potent competitive inhibitor of phenylalanine hydroxylase (Ki = 10(-6) M) and its accumulation in the epidermis of patients with vitiligo blocks the supply of L-tyrosine from L-phenylalanine. 4a-hydroxytetrahydrobiopterin dehydratase has a dual function as the activator/dimerization catalyst for the transcription factor hepatocyte nuclear factor I (HNF-I). HNF-I binds to a 16-base inverted palindrome which seems to be present on the promoters of both the tyrosinase and phenylethanolamine-N-methyl transferase (PNMT) genes. Therefore, defective 4a-hydroxytetrahydrobiopterin dehydratase in vitiligo influences not only the supply of L-tyrosine but also the transcription of the tyrosinase gene in melanocytes. Furthermore, a similar transcriptional regulation of the PNMT gene in keratinocytes offers a possible explanation for the accumulation of norepinephrine in these patients.
Assuntos
Biopterinas/análogos & derivados , Catecolaminas/biossíntese , Pele/metabolismo , Vitiligo/metabolismo , Sequência de Bases , Biopterinas/biossíntese , Catecolaminas/sangue , Catecolaminas/urina , GTP Cicloidrolase/análise , Humanos , Hidroliases/análise , Queratinócitos/metabolismo , Melaninas/biossíntese , Melanócitos/metabolismo , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Fenilalanina Hidroxilase/análise , Fenilalanina Hidroxilase/antagonistas & inibidores , Fenilalanina Hidroxilase/genética , Feniletanolamina N-Metiltransferase/análise , Pterinas/metabolismo , Pele/químicaRESUMO
Recent studies indicate that membrane-associated thioredoxin reductase (TR) is a possible regulator of melanin biosynthesis via the inhibition of tyrosinase by reduced thioredoxin. In normal individuals, the levels of TR activity in skin correlate linearly to the Fitzpatrick classification of skin type, being lowest in type I skin and highest in skin type VI. In this study, TR was measured in 3-mm skin biopsies in Hermansky-Pudlak syndrome (HPS) patients and their relatives. Forty-five individuals from seven Puerto Rican kindreds were tested, including 12 homozygotes, nine obligate heterozygotes, and 24 unclassified individuals. In addition, seven separate nonkindred HPS patients were tested. With one exception, TR activity was markedly decreased in 18 homozygotes. TR activity was decreased in eight obligate heterozygotes and in 12 unclassified kindred members, whereas 10 subjects had normal TR activity when compared to the expected activity of their skin type. Four individuals were excluded from the analysis because of inadequate controls for their age group or immunosuppressive treatment for kidney transplant. The results indicate that decreased TR activity assayed in 3-mm skin punch biopsies is a useful method for detecting carriers of the HPS gene.
Assuntos
Albinismo/enzimologia , Triagem de Portadores Genéticos , NADH NADPH Oxirredutases/análise , Tiorredoxina Dissulfeto Redutase/análise , Adolescente , Adulto , Albinismo/genética , Transtornos Plaquetários/enzimologia , Cálcio/análise , Ceroide/metabolismo , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pele/enzimologia , SíndromeRESUMO
Suction blister roofs taken from the involved and uninvolved epidermis of patients with vitiligo showed a consistent reduction in levels of catalase compared to normal healthy controls of matched photo-skin types (Fitzpatrick classification). A decrease in catalase activity is expected to increase the concentration of hydrogen peroxide in the epidermis of these patients. Hydrogen peroxide functions as a reversible inhibitor of human tyrosinase with a KI of 8 X 10(-6) M. Also, hydrogen peroxide undergoes photochemical reduction yielding highly reactive hydroxyl radicals (OH.) and hydroxyl ions (OH-) mainly by the Haber-Weiss reaction. Hydroxyl radicals are capable of bleaching constitutional melanin and cause membrane lysis through lipid peroxidation reactions. Hydroxyl ions increase the pH in the epidermis, and as a consequence glutathione reductase activity is increased in patients with vitiligo compared to controls. Based on these new results, together with the previously reported calcium transport defect, a new hypothesis has been formulated for the pathogenesis of vitiligo.
Assuntos
Catalase/análise , Pele/enzimologia , Vitiligo/enzimologia , Glutationa Redutase/análise , Humanos , Pele/citologia , Pele/ultraestrutura , Tiorredoxina Dissulfeto Redutase/análiseRESUMO
Cell cultures of human keratinocytes contain membrane-associated thioredoxin reductase that is extremely active in reducing radicals on the outer plasma membrane. This enzyme activity was confirmed by its purification from cultures of stratified human keratinocytes by affinity column chromatography. The enzyme was assayed both in vivo and in vitro using a spin-labeled quaternary ammonium compound as the substrate, under saturating conditions in free radical substrate. Specific activities were determined by monitoring the sequential decrease in the amplitude of the electron spin resonance signal per unit of cell protein. The following properties were found: Cultures of adult stratified cells have approximately twice the thioredoxin reductase activity of neonatal cells. The enzyme is inhibited by thioprotein inhibitors (i.e., parachloromecuribenzoate and dinitrochlorobenzene). The activity is regulated by calcium concentrations of the cell culture medium. Stratified keratinocytes are half as active in medium containing 2 mM Ca++ compared with 0.1 mM Ca++ concentration. Product inhibition of the enzyme occurs with oxidized coenzyme NADP+ (i.e., 87% inhibition of enzyme activity over 30 min). The enzyme is heat stable at temperatures of 70 degrees C for 10 min. It is inactivated at 75 degrees C. A comparative study of thioredoxin reductase activity on stratified differentiated and undifferentiated rapidly growing cells was performed. Also, enzyme activity was quantitated for cultured keratinocytes isolated from vitiliginous and normal skin of the same donor. The results of this study, and the connection between this enzyme activity and UV-generated free radicals are reconciled in terms of the mechanism of action and metabolic activity of thioredoxin reductase.