RESUMEN
Vitiligo is characterized by a mostly progressive loss of the inherited skin color. The cause of the disease is still unknown, despite accumulating in vivo and in vitro evidence of massive oxidative stress via hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)) in the skin of affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Since depletion of the essential amino acid L-tryptophan (Trp) severely affects various immune responses, we here looked at Trp metabolism and signaling in these patients. Our in vivo and in vitro data revealed total absence of epidermal Trp hydroxylase activities and the presence of H(2)O(2)/ONOO(-) deactivated indoleamine 2,3-dioxygenase. Aryl hydrocarbon receptor signaling is severely impaired despite the ligand (Trp dimer) being formed, as shown by mass spectrometry. Loss of this signal is supported by the absence of downstream signals (COX-2 and CYP1A1) as well as regulatory T-lymphocytes and by computer modeling. In vivo Fourier transform Raman spectroscopy confirmed the presence of Trp metabolites together with H(2)O(2) supporting deprivation of the epidermal Trp pool by Fenton chemistry. Taken together, our data support a long-expressed role for in loco redox balance and a distinct immune response. These insights could open novel treatment strategies for this disease.
Asunto(s)
Epidermis/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/metabolismo , Vitíligo/metabolismo , Simulación por Computador , Humanos , Peróxido de Hidrógeno/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Estrés Oxidativo , Ácido Peroxinitroso/metabolismo , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Triptófano/metabolismo , Vitíligo/inmunologíaRESUMEN
Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the hydroxylation of hypoxanthine to xanthine and finally to uric acid in purine degradation. These reactions generate H(2)O(2) yielding allantoin from uric acid when reactive oxygen species accumulates. The presence of XO in the human epidermis has not been shown so far. As patients with vitiligo accumulate H(2)O(2) up to mm levels in their epidermis, it was tempting to examine whether this enzyme and consequently allantoin contribute to the oxidative stress theory in this disease. To address this question, reverse transcription-polymerase chain reaction, immunoreactivity, western blot, enzyme kinetics, computer modelling and high performance liquid chromatography/mass spectrometry analysis were carried out. Our results identified the presence of XDH/XO in epidermal keratinocytes and melanocytes. The enzyme is regulated by H(2)O(2) in a concentration-dependent manner, where concentrations of 10(-6 )m upregulates the activity. Moreover, we demonstrate the presence of epidermal allantoin in acute vitiligo, while this metabolite is absent in healthy controls. H(2)O(2)-mediated oxidation of Trp and Met in XO yields only subtle alterations in the enzyme active site, which is in agreement with the enzyme kinetics in the presence of 10(-3 )m H(2)O(2). Systemic XO activities are not affected. Taken together, our results provide evidence that epidermal XO contributes to H(2)O(2)-mediated oxidative stress in vitiligo via H(2)O(2)-production and allantoin formation in the epidermal compartment.
Asunto(s)
Queratinocitos/enzimología , Melanocitos/enzimología , Estrés Oxidativo , Vitíligo/metabolismo , Xantina Deshidrogenasa/metabolismo , Xantina Oxidasa/metabolismo , Alantoína/biosíntesis , Western Blotting , Estudios de Casos y Controles , Dominio Catalítico , Células Cultivadas , Simulación por Computador , Epidermis/metabolismo , Flavina-Adenina Dinucleótido/análogos & derivados , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Inmunohistoquímica , Modelos Químicos , Estructura Molecular , Oxidación-Reducción , ARN Mensajero/metabolismo , Ácido Úrico/metabolismoRESUMEN
The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5x10(-3)M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. (45)Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H(2)O(2)-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8m(2) surface area with its calcium gradient in the 10(-3)M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue.
Asunto(s)
Acetilcolinesterasa/metabolismo , Butirilcolinesterasa/metabolismo , Calcio/farmacología , Neurotoxinas/farmacología , Organofosfatos/farmacología , Piel/efectos de los fármacos , Piel/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Butirilcolinesterasa/química , Inhibidores de la Colinesterasa , Simulación por Computador , Motivos EF Hand , Activación Enzimática/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Hidrólisis , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
The human epidermis holds the capacity for autocrine cholinergic signal transduction, but the presence of butyrylcholinesterase (BchE) has not been shown so far. Our results demonstrate that this compartment transcribes a functional BchE. Its activity is even higher compared to acetylcholinesterase (AchE). Moreover, we show that BchE is subject to regulation by H(2)O(2) in a concentration-dependent manner as it was recently described for AchE. Epidermal BchE protein expression and enzyme activities are severely affected by H(2)O(2) in vitiligo as previously demonstrated for AchE. Removal/reduction of H(2)O(2) by a pseudocatalase PC-KUS yields normal/increased protein expression and activities. H(2)O(2)-mediated oxidation of methionine residues in BchE was confirmed by FT-Raman spectroscopy. Computer simulation supported major alteration of the enzyme active site and its tetramerisation domain suggesting deactivation of the enzyme due to H(2)O(2)-mediated oxidation. Based on our results we conclude that H(2)O(2) is a major player in the regulation of the cholinergic signal via both AchE and BchE and this signal is severely affected in the epidermis of patients with active vitiligo.
Asunto(s)
Butirilcolinesterasa/metabolismo , Epidermis/metabolismo , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo , Vitíligo/metabolismo , Acetilcolinesterasa/metabolismo , Sitios de Unión , Butirilcolinesterasa/química , Butirilcolinesterasa/genética , Células Cultivadas , Simulación por Computador , Regulación Enzimológica de la Expresión Génica , Humanos , Peróxido de Hidrógeno/farmacología , Queratinocitos/enzimología , Melanocitos/enzimología , Metionina/análogos & derivados , Metionina/metabolismo , Modelos Moleculares , Oxidación-Reducción/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/genética , Sulfóxidos/metabolismo , Vitíligo/patologíaRESUMEN
The human epidermis holds the full machinery for cholinergic signal transduction. However, the presence of the vesicular transporter (vesicular acetylcholine (ACh) transporter (VAChT)) for both choline and ACh has never been shown in this compartment. The results of this study confirm the presence of VAChT in cutaneous nerves and in both epidermal melanocytes and keratinocytes as well as in their nuclei using immunofluorescence labelling in situ and in vitro, Western blot analysis of cellular and nuclear extracts and reverse transcription-PCR. These results underline that ACh/choline transport in the non-neuronal epidermis is no different from the neuronal pathway. However, the function of VAChT in the nucleus remains to be shown.
Asunto(s)
Epidermis/metabolismo , Queratinocitos/metabolismo , Melanocitos/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Acetilcolina/metabolismo , Especificidad de Anticuerpos , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Fibras Colinérgicas/metabolismo , Células Epidérmicas , Epidermis/inervación , Humanos , Queratinocitos/citología , Melanocitos/citología , ARN Mensajero/análisis , Proteínas de Transporte Vesicular de Acetilcolina/genética , Proteínas de Transporte Vesicular de Acetilcolina/inmunologíaRESUMEN
To date there is ample in vivo and in vitro evidence for increased epidermal and systemic hydrogen peroxide (H(2)O(2)) levels in vitiligo, which can be reduced with a topical application of a pseudocatalase-K.U. Schallreuter (PC-KUS) leading to the recovery of epidermal catalase levels as well as other enzymes in peripheral blood cells. Recently, the generation of H(2)O(2) by oxidative metabolism of estrogens and other aromatic steroids was documented. Therefore, it was tempting to follow estrogen-generated H(2)O(2) and its possible effect on DNA damage in peripheral blood lymphocytes from patients with vitiligo before and after the reduction of epidermal H(2)O(2) with pseudocatalase PC-KUS compared to controls. For this purpose, 20 Caucasian patients were grouped in treated responders (group A, n=11) and untreated active/acute disease (group B, n=9) and compared to Caucasian healthy controls (group C, n=7). Consequently, epidermal catalase protein expression in full skin biopsies was assessed using immunofluorescence labelling together with determination of basal H(2)O(2) levels in peripheral blood lymphocytes. To test the influence of estrogen on H(2)O(2) generation and DNA damage, freshly prepared peripheral blood lymphocytes from all three groups were used for the alkaline comet assay in the presence and absence of catalase. The results of this study demonstrated that reduction of epidermal H(2)O(2) leads to both increased epidermal catalase protein expression as well as decreased H(2)O(2) concentrations in lymphocytes. Moreover, a direct estrogen-mediated DNA damage was identified in both patient groups, which was absent in healthy controls. This effect was not abolished by catalase pointing to direct quinone-mediated DNA damage by estrogens in peripheral blood lymphocytes in vitiligo.
Asunto(s)
Daño del ADN , Estrógenos/toxicidad , Peróxido de Hidrógeno/metabolismo , Linfocitos/metabolismo , Quinonas/toxicidad , Vitíligo/metabolismo , Adulto , Catalasa/metabolismo , Catalasa/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés OxidativoRESUMEN
Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3)M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6)M) activate this enzyme by increasing the Vmax>2-fold, meanwhile high concentrations of H2O2 (10(-3)M) inhibit the enzyme with a significant decrease in Vmax. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambdamax 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo.