RESUMEN
Absence of a functional proteasome in the suprabasal layers of the epidermis is responsible for keratosis linearis with ichthyosis congenital and sclerosing keratoderma syndrome. Patient epidermis shows hypergranulosis associated with abnormally shaped keratohyalin granules and abnormal distribution of filaggrin in the Stratum granulosum and Stratum corneum. This suggests that the proteasome is involved in the degradation of filaggrin. To test this hypothesis, the proteasome proteolytic activity was inhibited in 3D reconstructed human epidermis (RHE) with the specific clasto-lactacystin ß-lactone inhibitor. Confirming the efficacy of inhibition, ubiquitinated proteins accumulated in treated RHEs as compared to controls. Levels of urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), the end products of filaggrin degradation, were reduced. However, neither filaggrin accumulation nor appearance of filaggrin-derived peptides were observed. On the contrary, the amount of filaggrin was shown to decrease, and a similar tendency was observed for profilaggrin, its precursor. Accumulation of small cytoplasmic vesicles associated with a significant increase in autophagy markers indicated activation of the autophagy process upon proteasome inhibition. Taken together, these results suggest that the perturbation of UCA and PCA production after proteasome inhibition was probably due to down-regulation of filaggrin expression rather than to blocking of filaggrin proteolysis.
Asunto(s)
Proteínas Filagrina , Complejo de la Endopetidasa Proteasomal , Humanos , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
BACKGROUND: Central centrifugal cicatricial alopecia (CCCA) is the most common form of scarring alopecia among women of African ancestry. The disease is occasionally observed to affect women in families in a manner that suggests an autosomal dominant trait and usually manifests clinically after intense hair grooming. We sought to determine whether there exists a genetic basis of CCCA and, if so, what it is. METHODS: We used exome sequencing in a group of women with alopecia (discovery set), compared the results with those in a public repository, and applied other filtering criteria to identify candidate genes. We then performed direct sequencing to identify disease-associated DNA variations and RNA sequencing, protein modeling, immunofluorescence staining, immunoblotting, and an enzymatic assay to evaluate the consequences of potential etiologic mutations. We used a replication set that consisted of women with CCCA to confirm the data obtained with the discovery set. RESULTS: In the discovery set, which included 16 patients, we identified one splice site and three heterozygous missense mutations in PADI3 in 5 patients (31%). (The approximate prevalence of the disease is up to 5.6%.) PADI3 encodes peptidyl arginine deiminase, type III (PADI3), an enzyme that post-translationally modifies other proteins that are essential to hair-shaft formation. All three CCCA-associated missense mutations in PADI3 affect highly conserved residues and are predicted to be pathogenic; protein modeling suggests that they result in protein misfolding. These mutations were found to result in reduced PADI3 expression, abnormal intracellular localization of the protein, and decreased enzymatic activity - findings that support their pathogenicity. Immunofluorescence staining showed decreased expression of PADI3 in biopsy samples of scalp skin obtained from patients with CCCA. We then directly sequenced PADI3 in an additional 42 patients (replication set) and observed genetic variants in 9 of them. A post hoc analysis of the combined data sets showed that the prevalence of PADI3 mutation was higher among patients with CCCA than in a control cohort of women of African ancestry (P = 0.002 by the chi-square test; P = 0.006 by Fisher's exact test; and after adjustment for relatedness of persons, P = 0.03 and P = 0.04, respectively). CONCLUSIONS: Mutations in PADI3, which encodes a protein that is essential to proper hair-shaft formation, were associated with CCCA. (Funded by the Ram Family Foundation and others.).
Asunto(s)
Alopecia/genética , Negro o Afroamericano/genética , Predisposición Genética a la Enfermedad , Cabello/crecimiento & desarrollo , Mutación , Desiminasas de la Arginina Proteica/genética , Adolescente , Adulto , Edad de Inicio , Alopecia/etnología , Distribución de Chi-Cuadrado , Cicatriz/genética , Exoma , Femenino , Heterocigoto , Humanos , Persona de Mediana Edad , Mutagénesis , Linaje , Arginina Deiminasa Proteína-Tipo 3 , Desiminasas de la Arginina Proteica/metabolismo , Cuero Cabelludo/patología , Análisis de Secuencia de ADNRESUMEN
The discovery in 2006 that loss-of-function mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris and can predispose to atopic dermatitis (AD) galvanized the dermatology research community and shed new light on a skin protein that was first identified in 1981. However, although outstanding work has uncovered several key functions of filaggrin in epidermal homeostasis, a comprehensive understanding of how filaggrin deficiency contributes to AD is still incomplete, including details of the upstream factors that lead to the reduced amounts of filaggrin, regardless of genotype. In this review, we re-evaluate data focusing on the roles of filaggrin in the epidermis, as well as in AD. Filaggrin is important for alignment of keratin intermediate filaments, control of keratinocyte shape, and maintenance of epidermal texture via production of water-retaining molecules. Moreover, filaggrin deficiency leads to cellular abnormalities in keratinocytes and induces subtle epidermal barrier impairment that is sufficient enough to facilitate the ingress of certain exogenous molecules into the epidermis. However, although FLG null mutations regulate skin moisture in non-lesional AD skin, filaggrin deficiency per se does not lead to the neutralization of skin surface pH or to excessive transepidermal water loss in atopic skin. Separating facts from chaff regarding the functions of filaggrin in the epidermis is necessary for the design efficacious therapies to treat dry and atopic skin.
Asunto(s)
Dermatitis Atópica , Ictiosis Vulgar , Dermatitis Atópica/genética , Dermatitis Atópica/metabolismo , Proteínas Filagrina , Humanos , Ictiosis Vulgar/genética , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Agua/metabolismoRESUMEN
Deimination (or citrullination) is a post-translational modification catalyzed by a calcium-dependent enzyme family of five peptidylarginine deiminases (PADs). Deimination is involved in physiological processes (cell differentiation, embryogenesis, innate and adaptive immunity, etc.) and in autoimmune diseases (rheumatoid arthritis, multiple sclerosis and lupus), cancers and neurodegenerative diseases. Intermediate filaments (IF) and associated proteins (IFAP) are major substrates of PADs. Here, we focus on the effects of deimination on the polymerization and solubility properties of IF proteins and on the proteolysis and cross-linking of IFAP, to finally expose some features of interest and some limitations of citrullinomes.
Asunto(s)
Artritis Reumatoide/enzimología , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/enzimología , Esclerosis Múltiple/enzimología , Neoplasias/enzimología , Enfermedades Neurodegenerativas/enzimología , Procesamiento Proteico-Postraduccional , Desiminasas de la Arginina Proteica/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Diferenciación Celular , Citrulinación , Células Epiteliales/enzimología , Células Epiteliales/patología , Proteínas Filagrina , Humanos , Proteínas de Filamentos Intermediarios/química , Proteínas de Filamentos Intermediarios/genética , Filamentos Intermedios/ultraestructura , Células Madre Mesenquimatosas/enzimología , Células Madre Mesenquimatosas/patología , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/inmunología , Enfermedades Neurodegenerativas/patología , Neuronas/enzimología , Neuronas/patología , Multimerización de Proteína , Desiminasas de la Arginina Proteica/química , Desiminasas de la Arginina Proteica/genética , Proteolisis , SolubilidadRESUMEN
Deimination, also known as citrullination, corresponds to the conversion of the amino acid arginine, within a peptide sequence, into the non-standard amino acid citrulline. This post-translational modification is catalyzed by a family of calcium-dependent enzymes called peptidylarginine deiminases (PADs). Deimination is implicated in a growing number of physiological processes (innate and adaptive immunity, gene regulation, embryonic development, etc.) and concerns several human diseases (rheumatoid arthritis, neurodegenerative diseases, female infertility, cancer, etc.). Here, we update the involvement of PADs in both the homeostasis of skin and skin diseases. We particularly focus on keratinocyte differentiation and the epidermal barrier function, and on hair follicles. Indeed, alteration of PAD activity in the hair shaft is responsible for two hair disorders, the uncombable hair syndrome and a particular form of inflammatory scarring alopecia, mainly affecting women of African ancestry.
Asunto(s)
Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional , Desiminasas de la Arginina Proteica/genética , Enfermedades de la Piel/genética , Fenómenos Fisiológicos de la Piel/genética , Citrulinación , Homeostasis/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Queratinocitos/metabolismo , Desiminasas de la Arginina Proteica/metabolismo , Piel/metabolismo , Piel/patología , Enfermedades de la Piel/metabolismoRESUMEN
Uncombable hair syndrome (UHS), also known as "spun glass hair syndrome," "pili trianguli et canaliculi," or "cheveux incoiffables" is a rare anomaly of the hair shaft that occurs in children and improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant to being combed flat. Until now, both simplex and familial UHS-affected case subjects with autosomal-dominant as well as -recessive inheritance have been reported. However, none of these case subjects were linked to a molecular genetic cause. Here, we report the identification of UHS-causative mutations located in the three genes PADI3 (peptidylarginine deiminase 3), TGM3 (transglutaminase 3), and TCHH (trichohyalin) in a total of 11 children. All of these individuals carry homozygous or compound heterozygous mutations in one of these three genes, indicating an autosomal-recessive inheritance pattern in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic causes of UHS and shed light on its pathophysiology and hair physiology in general.
Asunto(s)
Antígenos/genética , Enfermedades del Cabello/genética , Cabello/crecimiento & desarrollo , Hidrolasas/genética , Proteínas de Filamentos Intermediarios/genética , Mutación , Transglutaminasas/genética , Adolescente , Animales , Secuencia de Bases , Línea Celular , Codón sin Sentido , Femenino , Cabello/anomalías , Cabello/anatomía & histología , Cabello/metabolismo , Humanos , Hidrolasas/deficiencia , Hidrolasas/metabolismo , Masculino , Ratones , Ratones Noqueados , Modelos Moleculares , Mutación Missense/genética , Conformación Proteica , Arginina Deiminasa Proteína-Tipo 3 , Desiminasas de la Arginina Proteica , Transglutaminasas/deficiencia , Transglutaminasas/metabolismo , Vibrisas/anomalíasRESUMEN
Deimination or citrullination is a post-translational modification catalysed by a family of calcium-dependent enzymes called peptidylarginine deiminases (PADs). It corresponds to the transformation of arginine residues within a peptide sequence into citrulline residues. Deimination induces a decreased net charge of targeted proteins; therefore, it alters their folding and changes intra- and intermolecular ionic interactions. Deimination is involved in several physiological processes (inflammation, gene regulation, etc.) and human diseases (rheumatoid arthritis, neurodegenerative diseases, cancer, etc.). Here, we describe the PADs expressed in the epidermis and their known substrates, focusing on their role in the epidermal barrier function.
Asunto(s)
Epidermis/enzimología , Desiminasas de la Arginina Proteica/metabolismo , Animales , Arginina/metabolismo , Artritis Reumatoide/metabolismo , Calcio/metabolismo , Citrulina/metabolismo , Células Epidérmicas/metabolismo , Proteínas Filagrina , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Hidrolasas/metabolismo , Inflamación , Ratones , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The main function of the epidermis is to establish a vital multifunctional barrier between the body and its external environment. A defective epidermal barrier is one of the key features of atopic dermatitis (AD), a chronic and relapsing inflammatory skin disorder that affects up to 20% of children and 2-3% of adults and often precedes the development of allergic rhinitis and asthma. This review summarizes recent discoveries on the origin of the skin barrier alterations in AD at the structural protein level, including hereditary and acquired components. The consequences of the epidermal barrier alteration on our current understanding of the pathogenesis of AD, and its possible implications on the treatment of patients, are discussed here.
Asunto(s)
Córnea/metabolismo , Dermatitis Atópica/metabolismo , Epidermis/metabolismo , Proteínas/metabolismo , Animales , Córnea/patología , Citocinas/metabolismo , Dermatitis Atópica/patología , Epidermis/patología , HumanosRESUMEN
BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by a disturbed epidermal barrier. In a subset of patients, this is explained by nonsense mutations in the gene encoding filaggrin (FLG). OBJECTIVES: We sought to evaluate the respective role of FLG mutations and proinflammatory cytokines and to assess the expression of FLG, hornerin (HRNR), and FLG2, 2 FLG-like proteins, which are involved in epidermal barrier functions, in normal skin and both lesional and nonlesional skin of patients with AD. METHODS: An FLG-genotyped cohort of 73 adults with AD and 73 aged-matched control subjects was analyzed by using immunohistochemistry and immunoblotting. Normal primary human keratinocytes were differentiated in either the absence or presence of IL-4, IL-13, and IL-25. RESULTS: Compared with control subjects, FLG, HRNR, and FLG2 were detected at significantly lower levels in the skin of patients with AD, irrespective of their FLG genotype. The reduction was greater in lesional compared with nonlesional skin. In addition, the proFLG/FLG ratio was found to be higher in the skin of wild-type patients than in control subjects. Cytokine treatment of keratinocytes induced a dramatic reduction in FLG, FLG2, and HRNR expression both at the mRNA and protein levels. CONCLUSION: The stratum corneum of lesional but also clinically unaffected skin of adults with AD is abnormal, with reduced expression of FLG and FLG-like proteins. In addition to nonsense mutations, proinflammatory cytokines and some defects in the proFLG processing can contribute to the FLG downregulation. Our study suggests that skin inflammation reduces the expression of FLG-like proteins, contributing to the AD-related epidermal barrier dysfunction.
Asunto(s)
Proteínas de Unión al Calcio/genética , Dermatitis Atópica/genética , Proteínas de Filamentos Intermediarios/genética , Proteínas S100/genética , Piel/inmunología , Adulto , Western Blotting , Proteínas de Unión al Calcio/inmunología , Estudios de Casos y Controles , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Regulación hacia Abajo , Femenino , Proteínas Filagrina , Expresión Génica , Humanos , Inmunohistoquímica , Interleucina-13/farmacología , Interleucina-17/farmacología , Interleucina-4/farmacología , Proteínas de Filamentos Intermediarios/inmunología , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Queratinocitos/patología , Masculino , Cultivo Primario de Células , Proteínas S100/inmunología , Piel/patologíaRESUMEN
FLG is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it colocalized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis using benzyloxycarbonyl-pro-prolinal induced the accumulation of FLG monomers. Downregulation of PREP expression in reconstructed human epidermis using RNA interference confirmed the impact of PREP on FLG metabolism and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, western blotting, and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification such as loricrin. Consequently, at the functional level, the transepidermal electric resistance was drastically reduced.
RESUMEN
Peptidylarginine deiminases (PADs) transform a protein arginine residue into the non-standard amino acid citrulline. This calcium-dependent post-translational modification of proteins is called citrullination or deimination. As described in this special issue, PADs play a role in various physiological processes, and PAD deregulations are involved in many human diseases. Three PADs are expressed in the epidermis, where their roles begin to be deciphered. PAD1 and PAD3 are involved in keratinocyte differentiation, particularly in the epidermal barrier function, keratins, filaggrin and filaggrin-related proteins being the most abundant deiminated epidermal proteins. Reduced amounts of deiminated proteins and PAD1 expression may be involved in the pathogenesis of psoriasis and atopic dermatitis, two very frequent and chronic skin inflammatory diseases. The trichohyalin/PAD3/transglutaminase three pathway is important for hair shaft formation. Mutations of the PADI3 gene, leading to a decreased activity or abnormal localization of the corresponding isotype, are the cause of a rare hair disorder called uncombable hair syndrome, and are associated with the central centrifugal cicatricial alopecia, a frequent alopecia mainly affecting women of African ancestry. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.
Asunto(s)
Proteínas Filagrina , Cabello , Hidrolasas , Femenino , Humanos , Alopecia/metabolismo , Epidermis , Hidrolasas/genética , Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismoRESUMEN
Deimination is a post-translational modification catalyzed by a family of enzymes named peptidylarginine deiminases (PADs). PADs transform arginine residues of protein substrates into citrulline. Deimination has been associated with numerous physiological and pathological processes. In human skin, three PADs are expressed (PAD1-3). While PAD3 is important for hair shape formation, the role of PAD1 is less clear. To decipher the main role(s) of PAD1 in epidermal differentiation, its expression was down-regulated using lentivirus-mediated shRNA interference in primary keratinocytes and in three-dimensional reconstructed human epidermis (RHE). Compared to normal RHEs, down-regulation of PAD1 caused a drastic reduction in deiminated proteins. Whereas proliferation of keratinocytes was not affected, their differentiation was disturbed at molecular, cellular and functional levels. The number of corneocyte layers was significantly reduced, expression of filaggrin and cornified cell envelope components, such as loricrin and transglutaminases, was down-regulated, epidermal permeability increased and trans-epidermal-electric resistance diminished drastically. Keratohyalin granule density decreased and nucleophagy in the granular layer was disturbed. These results demonstrate that PAD1 is the main regulator of protein deimination in RHE. Its deficiency alters epidermal homeostasis, affecting the differentiation of keratinocytes, especially the cornification process, a special kind of programmed cell death.
RESUMEN
Filaggrin-2 (FLG2), a member of the S100-fused type protein family, shares numerous features with filaggrin (FLG), a key protein implicated in the epidermal barrier functions. Both display a related structural organization, an identical pattern of expression and localization in human epidermis, and proteolytic processing of a large precursor. Here, we tested whether FLG2 was a substrate of calpain 1, a calcium-dependent protease directly involved in FLG catabolism. In addition, deimination being critical for FLG degradation, we analyzed whether FLG2 deimination interfered with its proteolytic processing. With this aim, we first produced a recombinant form of FLG2 corresponding to subunits B7 to B10 fused to a COOH-terminal His tag. Incubation with calpain 1 in the presence of calcium induced a rapid degradation of the recombinant protein and the production of several peptides, as shown by Coomassie Blue-stained gels and Western blotting with anti-FLG2 or anti-His antibodies. MALDI-TOF mass spectrometry confirmed this result and further evidenced the production of non-immunoreactive smaller peptides. The degradation was not observed when a calpain 1-specific inhibitor was added. The calpain cleavage sites identified by Edman degradation were regularly present in the B-type repeats of FLG2. Moreover, immunohistochemical analysis of normal human skin revealed colocalization of FLG2 and calpain 1 in the upper epidermis. Finally, the FLG2 deiminated by human peptidylarginine deiminases was shown to be more susceptible to calpain 1 than the unmodified protein. Altogether, these data demonstrate that calpain 1 is essential for the proteolytic processing of FLG2 and that deimination accelerates this process.
Asunto(s)
Calcio/metabolismo , Calpaína/metabolismo , Epidermis/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Animales , Calcio/química , Calpaína/química , Calpaína/genética , Epidermis/química , Proteínas Filagrina , Humanos , Proteínas de Filamentos Intermediarios/química , Proteínas de Filamentos Intermediarios/genética , Ratones , Ratones Transgénicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Deimination, the conversion of protein-bound arginines into citrullines, is a post-translational modification catalyzed by a peptidylarginine deiminase (Pad). In the epidermis, three Pads are expressed, namely Pad1, 2 and 3, and the major deiminated protein is filaggrin. Deimination of fibrin has been observed in various pathological inflammatory conditions. Here, we analyzed the expression of Pads and citrullination of proteins during cutaneous wound healing, i.e. in a physiological inflammatory condition. Full-thickness punches were performed on adult mouse back skin, and wound recovery was analyzed over 10 days by immunohistology and western blotting. Pad1 was immunodetected in all the neo-epidermis. Pad3, normally expressed in the stratum granulosum, was not detected in the hyperproliferative tongue of the neo-epidermis, but was shown to be co-localized with (pro)filaggrin in a large number of keratinocyte layers in its differentiating part. Deiminated proteins were detected in the stratum corneum of the neo-epidermis in the late phase of re-epithelialization, and in the clot and the clot-derived scab. In the clot where we only detected Pad4, one of the deiminated proteins was shown to be fibrin. Deimination of the clot proteins, and more generally wound healing and keratinocyte differentiation, seemed to be Pad2-independent, as shown using Padi2(-/-) mice.
Asunto(s)
Hidrolasas/biosíntesis , Piel/lesiones , Cicatrización de Heridas/fisiología , Heridas y Lesiones/metabolismo , Animales , Biopsia , Western Blotting , Diferenciación Celular , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina Proteica , Piel/metabolismo , Piel/patología , Heridas y Lesiones/patologíaRESUMEN
Peptidylarginine deiminases (PADs) catalyze deimination, converting arginyl to citrullyl residues. Only three PAD isotypes are detected in the epidermis where they play a crucial role, targeting filaggrin, a key actor for the tissue hydration and barrier functions. Their expression and activation depends on the keratinocyte differentiation state. To investigate this regulation, we used primary keratinocytes induced to differentiate either by increasing cell-density or by treatment with vitamin D. High cell-density increased PAD1 and 3, but not PAD2, at the mRNA and protein levels, and up-regulated protein deimination. By contrast, vitamin D increased PAD1-3 mRNA amounts, with distinct kinetics, but neither the proteins nor the deimination rate. Furthermore, auto-deimination was shown to decrease PAD activity, increasing the distances between the four major amino acids of the active site. In summary, deimination can be regulated at multiple levels: transcription of the PADI genes, translation of the corresponding mRNAs, and auto-deimination of PADs.
Asunto(s)
Hidrolasas/metabolismo , Iminas/metabolismo , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Niño , Citrulina/química , Citrulina/metabolismo , Proteínas Filagrina , Humanos , Hidrolasas/genética , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Isoenzimas/genética , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/fisiología , Datos de Secuencia Molecular , Estructura Molecular , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vitamina D/farmacología , Vitaminas/farmacologíaRESUMEN
Deimination or citrullination, is a post-translational modification with many facets. It is involved in several basic cellular processes, including gene regulation, embryonic development and terminal differentiation, and also in various pathophysiological mechanisms linked to severe human diseases such as multiple sclerosis and rheumatoid arthritis. Deimination, the calcium-dependent enzymatic conversion of peptidyl-arginine to peptidyl-citrulline, induces a decrease in the charge of the modified proteins with major consequences on their conformation, stability and/or interactions, and therefore on their functions. Five isotypes of peptidylarginine deiminases (1-4 and 6), exist in humans with a variable tissue expression. These highly conserved enzymes are closely regulated at transcriptional and post-transcriptional levels, probably including auto-deimination.
Asunto(s)
Arginina/metabolismo , Citrulina/metabolismo , Hidrolasas/fisiología , Procesamiento Proteico-Postraduccional , Animales , Enfermedades Autoinmunes/enzimología , Agua Corporal/metabolismo , Calcio/fisiología , Cromatina/metabolismo , Secuencia Conservada , Córnea/metabolismo , Epidermis/enzimología , Evolución Molecular , Regulación de la Expresión Génica/fisiología , Humanos , Hidrolasas/deficiencia , Hidrolasas/genética , Isoenzimas/genética , Isoenzimas/fisiología , Ratones , Proteínas de la Mielina/metabolismo , Neoplasias/enzimología , Enfermedades del Sistema Nervioso/enzimología , Procesamiento Proteico-Postraduccional/fisiología , Arginina Deiminasa Proteína-Tipo 6 , Desiminasas de la Arginina Proteica , Enfermedades de la Piel/enzimologíaRESUMEN
Peptidylarginine deiminase (PAD, EC 3.5.3.15) enzyme catalyzes the conversion of arginine residues to citrulline residues in the presence of calcium ion, which is an elaborate post-translational modification on the target protein. Recently, five isoforms have been identified in mammals. Among them, three isoforms (type I, II, III) are expressed in the human epidermis, and involved in several skin physiological and pathological processes. In the past few years, several researches concerning the transcriptional regulation of three human PADI type genes (PADI1, PADI2 and PADI3) in the epidermis have been carried out. In this review, we describe an overview of the current outcomes about these studies with their significance. It is anticipated that these investigations will provide novel therapeutic and prophylactic targets for future approaches to the treatment or prevention of severe psoriasis and bullous congenital ichthyosiform erythroderma.
Asunto(s)
Hidrolasas/genética , Hidrolasas/metabolismo , Queratinocitos/metabolismo , Transcripción Genética/fisiología , Secuencia de Bases , Homeostasis/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Queratinocitos/citología , Datos de Secuencia Molecular , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina Proteica , Fenómenos Fisiológicos de la Piel/genéticaRESUMEN
Deimination, a post-translational modification catalyzed by a family of enzymes called peptidylarginine deiminases (PADs), is the conversion of arginine into citrulline residues in a protein. Deimination has been associated with numerous physiological and pathological processes. Our aim was to study its implication in the homeostasis of human epidermis, where three PADs are expressed, namely PAD1, 2, and 3. Three-dimensional reconstructed human epidermis (RHEs) were treated for 2 days with increased concentrations (0-800 µM) of Cl-amidine, a specific PAD inhibitor. Cl-amidine treatments inhibited deimination in a dose-dependent manner and were not cytotoxic for keratinocytes. At 800 µM , Cl-amidine was shown to reduce deimination by half, alter keratinocyte differentiation, decrease the number of corneocyte layers, significantly increase the number of transitional cells, induce clustering of mitochondria and of heterogeneous vesicles in the cytoplasm of granular keratinocytes, and upregulate the expression of autophagy proteins, including LC3-II, sestrin-2, and p62/SQSTM1. LC3 and PADs were further shown to partially co-localize in the upper epidermis. These results demonstrated that Cl-amidine treatments slow down cornification and alter autophagy in the granular layer. They suggest that PAD1 and/or PAD3 play a role in the constitutive epidermal autophagy process that appears as an important step in cornification.
Asunto(s)
Autofagia/fisiología , Epidermis/fisiología , Ornitina/análogos & derivados , Arginina Deiminasa Proteína-Tipo 1/metabolismo , Arginina Deiminasa Proteína-Tipo 3/metabolismo , Arginina/metabolismo , Autofagia/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Citrulina/metabolismo , Epidermis/efectos de los fármacos , Epidermis/ultraestructura , Humanos , Queratinocitos , Microscopía Electrónica de Transmisión , Ornitina/farmacología , Cultivo Primario de Células , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/fisiología , Arginina Deiminasa Proteína-Tipo 1/antagonistas & inhibidores , Arginina Deiminasa Proteína-Tipo 3/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
Human peptidylarginine deiminase type III gene (PADI3) encodes a crucial post-translational modification enzyme that converts protein-bound arginine residues into citrulline residues. Its expression is restricted to a few cell types, including keratinocytes in the granular layer of the epidermis and in the inner root sheath of hair follicles. In these cells, the enzyme is involved in terminal processing of intermediate filament-binding proteins such as filaggrin and trichohyalin. To study the molecular mechanisms that control the expression of PADI3 in human keratinocytes at the transcriptional level, we characterized its promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI3 coupled to the luciferase gene. We found that as few as 129 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that NF-Y (nuclear factor Y) and Sp1/Sp3 (specificity protein 1/3) bind to this region in vitro and in vivo. Moreover, mutation of the Sp1- or NF-Y-binding motif markedly reduced PADI3 promoter activity. Furthermore, Sp1 or NF-YA (NF-Y subunit) small interfering RNAs effectively diminished PADI3 expression in keratinocytes cultured in both low- and high-calcium medium. These data indicate that PADI3 expression is driven by Sp1/Sp3 and NF-Y binding to the promoter region.