RESUMO
PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.
Assuntos
Melanoma , Nevo , Neoplasias Cutâneas , Humanos , Imuno-Histoquímica , Melanoma/genética , Fenótipo , Neoplasias Cutâneas/genéticaAssuntos
Queratinócitos/imunologia , Biologia Molecular , Terapia de Alvo Molecular/métodos , Nevo Sebáceo de Jadassohn/genética , Nevo Sebáceo de Jadassohn/terapia , Biópsia , Proteínas Adaptadoras de Sinalização CARD/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Conexina 43/genética , Feminino , Guanilato Ciclase/genética , Humanos , Lactente , Inflamação , Queratinócitos/metabolismo , Masculino , Proteínas de Membrana/genética , Mosaicismo , Mutação de Sentido Incorreto , Resultado do TratamentoRESUMO
Keratitis-ichthyosis-deafness (KID) syndrome is a severe, untreatable condition characterized by ocular, auditory, and cutaneous abnormalities, with major complications of infection and skin cancer. Most cases of KID syndrome (86%) are caused by a heterozygous missense mutation (c.148G>A, p.D50N) in the GJB2 gene, encoding gap junction protein Cx26, which alters gating properties of Cx26 channels in a dominant manner. We hypothesized that a mutant allele-specific small interfering RNA could rescue the cellular phenotype in patient keratinocytes (KCs). A KID syndrome cell line (KID-KC) was established from primary patient KCs with a heterozygous p.D50N mutation. This cell line displayed impaired gap junction communication and hyperactive hemichannels, confirmed by dye transfer, patch clamp, and neurobiotin uptake assays. A human-murine chimeric skin graft model constructed with KID-KCs mimicked patient skin in vivo, further confirming the validity of these cells as a model. In vitro treatment with allele-specific small interfering RNA led to robust inhibition of the mutant GJB2 allele without altering expression of the wild-type allele. This corrected both gap junction and hemichannel activity. Notably, allele-specific small interfering RNA treatment caused only low-level off-target effects in KID-KCs, as detected by genome-wide RNA sequencing. Our data provide an important proof-of-concept and model system for the potential use of allele-specific small interfering RNA in treating KID syndrome and other dominant genetic conditions.
Assuntos
Conexinas/genética , Queratinócitos/fisiologia , Ceratite/genética , Mutação de Sentido Incorreto/genética , RNA Interferente Pequeno/genética , Pele/metabolismo , Alelos , Animais , Linhagem Celular , Quimera , Conexina 26 , Junções Comunicantes/metabolismo , Xenoenxertos , Heterozigoto , Humanos , Ceratite/terapia , Potenciais da Membrana , Camundongos , Pele/patologia , Transplante de PeleRESUMO
Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genodermatosis caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils at the dermal-epidermal junction. With no curative treatments presently available, retrovirally transduced autologous epidermal grafts and intradermal lentivirally engineered fibroblast injections are being investigated. Alternative approaches aim to infuse allogeneic mesenchymal stromal cells (MSCs) to provide a more generalized treatment for RDEB. We investigated whether healthy human MSCs could be engineered to overexpress C7 and correct RDEB in a human:murine chimeric model. Initially, engineered MSCs incorporated ex vivo into RDEB grafts, their presence confirmed by fluorescence in situ hybridization, revealed recovery of function of the dermal-epidermal junction with no signs of blister formation. Importantly, the detection of anchoring fibrils by transmission electron microscopy corroborated structural recovery. Next, MSCs cotransduced to express C7 and luciferase were delivered intradermally into grafted RDEB skin, resulting in localized MSC persistence with deposition of de novo C7 at the site. Notably, C7 expression was sufficient to restore anchoring fibril density to normal levels. In contrast, intravenously injected engineered MSCs were undetectable within grafts and lacked anchoring fibril reconstitution. Our data suggest that although localized correction may be achievable using engineered MSCs, strategies for systemic administration require further modeling.
Assuntos
Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/metabolismo , Células-Tronco Mesenquimais/fisiologia , Reticulina/metabolismo , Pele/patologia , Animais , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Engenharia Genética , Humanos , Camundongos , Camundongos SCID , Microscopia Eletrônica de Transmissão , Mutação/genética , Reticulina/ultraestrutura , Transplante de Pele , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Quimeras de TransplanteRESUMO
Loss-of-function mutations in the common gamma (γc) chain cytokine receptor subunit give rise to severe combined immunodeficiency characterized by lack of T and natural killer cells and infant death from infection. Hematopoietic stem cell transplantation or gene therapy offer a cure, but despite successful replacement of lymphoid immune lineages, a long-term risk of severe cutaneous human papilloma virus infections persists, possibly related to persistent γc-deficiency in other cell types. Here we show that keratinocytes, the only cell type directly infected by human papilloma virus, express functional γc and its co-receptors. After stimulation with the γc-ligand IL-15, γc-deficient keratinocytes show significantly impaired secretion of specific chemokines including CXCL1, CXCL8, and CCL20, resulting in reduced chemotaxis of dendritic cells and CD4+ T cells. Furthermore, γc-deficient keratinocytes also exhibit defective induction of T-cell chemotaxis in a model of stable human papilloma virus-18 infection. These findings suggest that persistent γc-deficiency in keratinocytes alters immune cell recruitment to the skin, which may contribute to the development and persistence of warts in this condition and would require different treatment approaches.
Assuntos
Quimiocinas/genética , Regulação da Expressão Gênica , Doença das Cadeias Pesadas/imunologia , Imunidade Inata , Cadeias gama de Imunoglobulina/metabolismo , Queratinócitos/metabolismo , Linfócitos T/imunologia , Linhagem Celular , Movimento Celular , Quimiocinas/biossíntese , Citometria de Fluxo , Doença das Cadeias Pesadas/genética , Doença das Cadeias Pesadas/metabolismo , Humanos , Queratinócitos/imunologia , Queratinócitos/patologia , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Filaggrin, which is encoded by the filaggrin gene (FLG), is an important component of the skin's barrier to the external environment, and genetic defects in FLG strongly associate with atopic dermatitis (AD). However, not all patients with AD have FLG mutations. OBJECTIVE: We hypothesized that these patients might possess other defects in filaggrin expression and processing contributing to barrier disruption and AD, and therefore we present novel therapeutic targets for this disease. RESULTS: We describe the relationship between the mechanistic target of rapamycin complex 1/2 protein subunit regulatory associated protein of the MTOR complex 1 (RAPTOR), the serine/threonine kinase V-Akt murine thymoma viral oncogene homolog 1 (AKT1), and the protease cathepsin H (CTSH), for which we establish a role in filaggrin expression and processing. Increased RAPTOR levels correlated with decreased filaggrin expression in patients with AD. In keratinocyte cell cultures RAPTOR upregulation or AKT1 short hairpin RNA knockdown reduced expression of the protease CTSH. Skin of CTSH-deficient mice and CTSH short hairpin RNA knockdown keratinocytes showed reduced filaggrin processing, and the mouse had both impaired skin barrier function and a mild proinflammatory phenotype. CONCLUSION: Our findings highlight a novel and potentially treatable signaling axis controlling filaggrin expression and processing that is defective in patients with AD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Catepsina H/metabolismo , Dermatite Atópica/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Western Blotting , Catepsina H/deficiência , Dermatite Atópica/patologia , Proteínas Filagrinas , Imunofluorescência , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Proteína Regulatória Associada a mTOR , Pele/metabolismo , Pele/patologiaRESUMO
Understanding the factors that influence N â S acyl transfer in native peptide sequences, and discovery of new reagents that facilitate it, will be key to expanding its scope and applicability. Here, through a study of short model peptides in thioester formation and cyclisation reactions, we demonstrate that a wider variety of Xaa-Cys motifs than originally envisaged are capable of undergoing efficient N â S acyl transfer. We present data for the relative rates of thioester formation and cyclisation for a representative set of amino acids, and show how this expanded scope can be applied to the production of the natural protease inhibitor Sunflower Trypsin Inhibitor-1 (SFTI-1).
Assuntos
Nitrogênio/química , Peptídeos/química , Enxofre/química , Acilação , Motivos de Aminoácidos , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Ciclização , Cisteína/química , Ésteres/química , Mercaptoetanol/química , Dados de Sequência Molecular , Peptídeos Cíclicos/química , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Netherton syndrome (NS) is a serious inherited skin disorder caused by mutations in the serine protease inhibitor Kazal type 5 gene (SPINK5), which encodes for a serine protease inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI). Patients with NS have defective keratinization, hair shaft defects, recurrent infections, atopy, and a predisposition to skin malignancies. Historically, 1 in 10 infants has died before their first birthday. Currently, there are no proven treatments to cure this condition. A SIN-lentiviral vector encoding the codon-optimized SPINK5 gene under the control of a 572 bp element derived from the human involucrin promoter can confer compartment-specific LEKTI expression in NS keratinocytes with restoration of normal skin architecture. Here we detail a study protocol for a phase I trial for feasibility and safety evaluations of autologous epidermal sheets generated from ex vivo gene-corrected keratinocyte stem cells, which will be grafted onto patients with mutation-proven NS.
Assuntos
Terapia Genética , Lentivirus/genética , Síndrome de Netherton/terapia , Ensaios Clínicos Fase I como Assunto/métodos , Feminino , Humanos , Queratinócitos/metabolismo , Queratinócitos/transplante , Masculino , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Inibidor de Serinopeptidase do Tipo Kazal 5RESUMO
Netherton syndrome (NS) is a serious inherited skin disorder caused by mutations in the gene SPINK5 (serine protease inhibitor Kazal type 5) which encodes for a serine protease inhibitor LEKTI (lymphoepithelial Kazal type-related inhibitor). Patients with NS have defective keratinization, hair shaft defects, recurrent infections, atopy and a predisposition to skin malignancies. Historically, one in ten infants has died before their first birthday. Currently there are no proven treatments to cure this condition. A SIN-lentiviral vector encoding the codon optimized SPINK5 gene under the control of a 572bp element derived from the human involucrin promoter (INVO) can confer compartment specific LEKTI expression in NS keratinocytes with restoration of normal skin architecture. Here we detail a study protocol for a phase I trial for feasibility and safety evaluations of autologous epidermal sheets generated from ex-vivo gene corrected keratinocyte stem cells, which will be grafted onto patients with mutation proven NS.
RESUMO
Lympho-Epithelial Kazal-Type-related Inhibitor (LEKTI) has been demonstrated to be an inhibitor of various kallikreins and is thought to play a role in the regulation of skin desquamation. In order to identify and investigate the potential of LEKTI to interact with other proteins, a method was developed using immobilised proteins onto arrays and nanoUPLC/MALDI-TOF MS. Using various domains of LEKTI, we demonstrated that these domains bound a number of kallikreins (5, 13 and 14) to varied extents on the array surface. Inhibitory assays confirmed that binding on the protein array surface corresponded directly to levels of inhibition. The method was then tested using skin epidermal extracts. All forms of rLEKTI with the exception of rLEKTI 12-15, demonstrated the binding of several potential candidate proteins. Surprisingly, the major binding partners of LEKTI were found to be the antimicrobial peptide dermcidin and the serine protease cathepsin G and no kallikreins. Using confocal microscopy and Netherton syndrome skin sections, we confirmed the co-localisation of LEKTI with dermcidin and demonstrated altered trafficking of dermcidin in these patients. This potential new role for LEKTI as a multifunctional protein in the protection and transport of proteins in the epidermis and its role in disease are discussed.
Assuntos
Catepsina G/metabolismo , Síndrome de Netherton/fisiopatologia , Peptídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/fisiologia , Inibidores de Serina Proteinase/metabolismo , Pele/metabolismo , Criança , Epiderme/metabolismo , Humanos , Calicreínas/metabolismo , Análise Serial de Proteínas , Ligação Proteica , Estrutura Terciária de Proteína , Inibidor de Serinopeptidase do Tipo Kazal 5 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Gene-modified skin grafts, produced through gene transfer to human keratinocyte stem cells, offer the possibility of therapeutic benefit for inherited skin diseases. We have previously described efficient lentiviral vector-mediated gene transfer to keratinocyte stem cells and the generation of human skin grafts for the inherited skin disease, Netherton syndrome, which arises due to mutations in serine protease inhibitor Kazal-type 5 (SPINK5). Vectors incorporating an internal murine retroviral-derived promoter [spleen focus-forming virus (SFFV)] in combination with a codon-optimized SPINK5 transgene supported high levels of reconstitution and robust correction of skin architecture. Subsequent longer-term experiments have uncovered unanticipated silencing phenomena, with loss of SPINK5 gene expression over time. The inadvertent introduction of CpG sites during codon optimization appears to have rendered vectors susceptible to silencing due to methylation across the promoter-transgene boundary. Substitution of the methylation-susceptible SFFV promoter with a 572-bp minimal human involucrin promoter (INVOp), which encodes very few CpG sites, prevented repression of the SPINK5 transgene and resulted in durable and highly compartment-specific reconstitution of lympho-epithelial Kazal-type-related inhibitor (LEKTI) in human skin grafted onto immunodeficient mice. We conclude that skin grafts modified with lentiviral vectors encoding INVOp offer a suitable platform for therapeutic gene therapy in Netherton syndrome, and our experience highlights unanticipated effects of transgene codon optimization.
Assuntos
Regulação da Expressão Gênica , Terapia Genética/métodos , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Transplante de Pele/métodos , Animais , Clonagem Molecular , Códon/genética , Ilhas de CpG , Metilação de DNA , Feminino , Citometria de Fluxo , Inativação Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Camundongos Nus , Precursores de Proteínas/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Inibidor de Serinopeptidase do Tipo Kazal 5 , Pele/citologia , Pele/metabolismo , TransgenesRESUMO
We performed genetic and immunohistochemical studies in a sister and brother with autosomal recessive neonatal inflammatory skin and bowel lesions. The girl died suddenly at 12 years of age from parvovirus B19-associated myocarditis; her brother had mild cardiomyopathy. We identified a loss-of-function mutation in ADAM17, which encodes a disintegrin and metalloproteinase 17 (also called tumor necrosis factor α [TNF-α]-converting enzyme, or TACE), as the probable cause of this syndrome. Peripheral-blood mononuclear cells (PBMCs) obtained from the brother at 17 years of age showed high levels of lipopolysaccharide-induced production of interleukin-1ß and interleukin-6 but impaired release of TNF-α. Despite repeated skin infections, this young man has led a relatively normal life. (Funded by Barts and the London Charity and the European Commission Seventh Framework Programme.).
Assuntos
Proteínas ADAM/genética , Doenças Inflamatórias Intestinais/genética , Deleção de Sequência , Dermatopatias/genética , Proteína ADAM17 , Adolescente , Criança , Evolução Fatal , Feminino , Humanos , Masculino , Miocardite/genética , Miocardite/virologia , LinhagemRESUMO
Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is recognized as a serine protease inhibitor and is thought to play a key role in skin barrier function through the inhibition of kallikrein (KLK) activities and regulation of skin desquamation. LEKTI has a total of 15 potential inhibitory domains, and we hypothesize that it has other potential targets in the skin. To identify candidate protease targets of LEKTI, a label-free quantitative proteomic approach was employed. This work describes a novel, rapid, and noninvasive method for the identification and quantitation of the major proteins present in the uppermost layers of the skin. By using cells scraped from the elbow, we were able to rapidly identify and quantitate 79 proteins. Caspase 14 and bleomycin hydrolase were identified as the proteases of highest abundance. Despite the fact that caspase 14 is a cysteine protease and LEKTI is described as a serine protease inhibitor, we demonstrate that caspase 14 is inhibited by full-length LEKTI and 5 recombinant fragments of LEKTI to varied extents. Details of the development of the methods used for the creation of the skin proteome and the inhibition of caspase 14 by LEKTI and implications for LEKTI as a multifunctional protease inhibitor are discussed.
Assuntos
Caspase 14/metabolismo , Epiderme/química , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas/análise , Proteômica/métodos , Inibidores de Serina Proteinase/metabolismo , Caspase 14/análise , Cromatografia Líquida , Cisteína Endopeptidases/análise , Humanos , Estrutura Terciária de Proteína , Inibidor de Serinopeptidase do Tipo Kazal 5 , Espectrometria de Massas em TandemRESUMO
Connexin 30 (Cx30) is a component of the gap junction complex. Dominant and recessive mutations in the GJB6 gene encoding Cx30 are associated with a variety of human inherited diseases primarily affecting the epidermis, hair, nail, and/or the inner ear. The underlying mechanism of disease associated with different GJB6 mutations such as the disruption of gap junction mediated intercellular communication is unknown. Towards understanding these disease mechanisms, transfection studies were performed in a keratinocyte cell line and in HeLa cells using EGFP tagged wildtype Cx30 and mutant Cx30 constructs harbouring dominant disease-associated GJB6 mutations. For all three of the skin disease-associated Cx30 mutations investigated, impaired trafficking of the protein to the plasma membrane was observed thus preventing the formation of functional Cx30 gap junctions. In contrast, the deafness-associated mutation T5M-Cx30/EGFP trafficked to the membrane but defective channel activity was observed following dye transfer studies.
Assuntos
Conexinas/genética , Surdez/genética , Mutação , Dermatopatias/genética , Sequência de Bases , Linhagem Celular , Membrana Celular/metabolismo , Conexina 30 , Conexinas/metabolismo , DNA/genética , Surdez/metabolismo , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Junções Comunicantes/metabolismo , Genes Dominantes , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Queratinócitos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fenótipo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Dermatopatias/metabolismoRESUMO
Distinct germline mutations in the gene (GJB3) encoding connexin 31 (Cx31) underlie the skin disease erythrokeratoderma variabilis (EKV) or sensorineural hearing loss with/without peripheral neuropathy. Here we describe a number of functional analyses to investigate the effect of these different disease-associated Cx31 mutants on connexon trafficking and intercellular communication. Immunostaining of a biopsy taken from an EKV patient harbouring the R42P mutation revealed sparse epidermal staining of Cx31, and, when present, it had a perinuclear localization. Transfection and microinjection studies in both keratinocytes and fibroblast cell lines also demonstrated that R42P and four other EKV-associated mutant Cx31 proteins displayed defective trafficking to the plasma membrane. The deafness/neuropathy only mutant 66delD had primarily a cytoplasmic localization, but some protein was visualized at the plasma membrane in a few transfected cells. Both 66delD- and R32W-Cx31/EGFP proteins had significantly impaired dye transfer rates compared to wild-type Cx31/EGFP protein. A striking characteristic feature observed with the dominant skin disease Cx31 mutations was a high incidence of cell death. This was not observed with wild-type, R32W 66delD Cx31 proteins. In conclusion, we have identified some key cellular phenotypic differences with respect to disease-associated Cx31 mutations.
Assuntos
Conexinas/genética , Ceratose/genética , Doenças do Sistema Nervoso Periférico/genética , Biópsia , Comunicação Celular/fisiologia , Morte Celular/fisiologia , Membrana Celular , Conexinas/metabolismo , Primers do DNA/química , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Fluorescência Verde , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Ceratose/metabolismo , Ceratose/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Doenças do Sistema Nervoso Periférico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Erythrokeratodermia variabilis (EKV) is a skin disorder characterized by variable (transient) erythemas and fixed keratosis. The disorder maps to chromosome 1p34-35, a location that contains the GJB3 gene encoding the gap junction protein connexin 31. Until now, only heterozygote mutations in the form of dominant inheritance have been described in this gene associated with EKV. We report here a homozygote mutation in the connexin 31 gene, found in a family that shows recessive inheritance of the disorder, thus providing the first molecular support for a recessive variant of EKV. The entire GJB3 coding sequence was scanned for mutations by sequencing. We detected a T-->C transition at position 101 of the coding sequence, which replaces a leucine with a proline at residue 34 of the protein (L34P). Evolutionary analysis shows that this mutation is located at a highly conserved region of connexin in the first putative transmembrane helix (TMH). In transfected keratinocytes, L34P connexin 31 had a cytoplasmic distribution, suggesting that the mutant form of this protein will not form normal gap junctions between adjacent cells. The change of leucine to proline is likely to alter the structure of the first TMH of connexin by inducing a kink, thus influencing connexon structure and function.