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1.
Nat Cell Biol ; 21(10): 1273-1285, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548606

RESUMO

Chromosome translocation is a major cause of the onset and progression of diverse types of cancers. However, the mechanisms underlying this process remain poorly understood. Here, we identified a non-homologous end-joining protein, IFFO1, which structurally forms a heterotetramer with XRCC4. IFFO1 is recruited to the sites of DNA damage by XRCC4 and promotes the repair of DNA double-strand breaks in a parallel pathway with XLF. Interestingly, IFFO1 interacts with lamin A/C, forming an interior nucleoskeleton. Inactivating IFFO1 or its interaction with XRCC4 or lamin A/C leads to increases in both the mobility of broken ends and the frequency of chromosome translocation. Importantly, the destruction of this nucleoskeleton accounts for the elevated frequency of chromosome translocation in many types of cancer cells. Our results reveal that the lamin A/C-IFFO1-constituted nucleoskeleton prevents chromosome translocation by immobilizing broken DNA ends during tumorigenesis.


Assuntos
Carcinogênese/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Lamina Tipo A/metabolismo , Translocação Genética , Animais , Carcinoma/genética , Cromossomos Humanos , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteínas de Filamentos Intermediários/genética , Camundongos , Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/fisiologia
2.
Forensic Sci Int ; 303: 109940, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31550598

RESUMO

The determination of cell type in biological casework samples would be helpful to identify the type of body fluids and interpret the DNA source in forensic laboratories. Exfoliated epidermal cells are considered to be a reasonable source of touch DNA; therefore, we developed and assessed an immunohistochemistry (IHC) procedure for identifying exfoliated epidermal cells as a screening test of touch DNA samples. Among five candidate protein markers investigated in this study, keratin 10 and kallikrein-related peptidase 5 were strongly expressed in the stratum corneum layer of the skin; however, their specificity was insufficient to identify epidermal cells. In contrast, IHC for corneodesmosin (CDSN), desmocollin 1 (DSC1), and filaggrin (FLG) was considered to be applicable because of their detectability and specificity on skin swab samples. Actually, CDSN and DSC1 could be good markers for exfoliated epidermal cells on touched contact traces that were contaminated with many unidentified impurities. Besides, positivity for FLG on mock casework samples appeared to be lower than for the other markers, which might be caused by its instability. Finally, the relationship between positivity for IHC and DNA yield was analyzed using skin swab samples. Although it was difficult to determine these correlations quantitatively because of the heterogeneous distribution of cells and the presence of cell-free DNA, the DNA-quantifiable samples analyzed in this study contained at least some of IHC-positive epidermal cells. In conclusion, IHC detection of skin-enriched proteins, especially CDSN and DSC1, could be useful for screening samples that have been handled or touched by someone before DNA analysis.


Assuntos
Desmocolinas/metabolismo , Células Epidérmicas/citologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pele/metabolismo , Tato , Biomarcadores/metabolismo , Análise Química do Sangue , Muco do Colo Uterino/química , DNA/análise , Ciências Forenses , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Calicreínas/metabolismo , Queratina-10/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Saliva/química , Sêmen/química , Coloração e Rotulagem
3.
Immunology ; 158(4): 281-286, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31509236

RESUMO

Despite sharing interleukin-4 receptor α (IL-4Rα) in their signaling cascades, IL-4 and IL-13 have different functions in atopic inflammation. IL-13 preferentially participates in the peripheral tissues because tissue-resident group 2 innate lymphoid cells produce IL-13 but not IL-4. In contrast, lymph node T follicular helper cells express IL-4 but not IL-13 to regulate B-cell immunity. The dominant microenvironment of IL-13 is evident in the lesional skin of atopic dermatitis (AD). The IL-13-rich local milieu causes barrier dysfunction by down-regulating the OVOL1-filaggrin (FLG) axis and up-regulating the periostin-IL-24 axis. Genome-wide association studies also point to the crucial involvement of the IL-13, OVOL1 and FLG genes in the pathogenesis of AD. Biologics targeting IL-13, such as the anti-IL-4Rα antibody dupilumab and the anti-IL-13 antibody tralokinumab, successfully improve AD lesions and further highlight the importance of IL-13 in the pathogenesis of AD.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Dermatite Atópica/imunologia , Interleucina-13/metabolismo , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Linfócitos/imunologia , Pele/imunologia , Fatores de Transcrição/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Terapia Biológica , Dermatite Atópica/terapia , Humanos , Imunidade Inata , Interleucina-13/imunologia , Subunidade alfa de Receptor de Interleucina-4/imunologia , Transdução de Sinais
4.
Int J Mol Sci ; 20(17)2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31480681

RESUMO

The main function of the skin is to protect the body from the external environment. The barrier function of the skin is mainly provided by the stratum corneum, which consists of corneocytes bound with the corneodesmosomes and lamellar lipids. Skin barrier proteins like loricrin and filaggrin also contribute to the skin barrier function. In various skin diseases, skin barrier dysfunction is a common symptom, and skin irritants like detergents or surfactants could also perturb skin barrier function. Many efforts have been made to develop strategies to improve skin barrier function. Here, we investigated whether the microfluidized lysates of Lactobacillus rhamnosus (LR), one of the most widely used probiotic species for various health benefits, may improve the skin barrier function in a reconstructed human epidermis, Keraskin™. Application of LR lysate on Keraskin™ increased the expression of tight junction proteins; claudin 1 and occludin as determined by immunofluorescence analysis, and skin barrier proteins; loricrin and filaggrin as determined by immunohistochemistry and immunofluorescence analysis and qPCR. Also, the cytotoxicity of a skin irritant, sodium lauryl sulfate (SLS), was alleviated by the pretreatment of LR lysate. The skin barrier protective effects of LR lysate could be further demonstrated by the attenuation of SLS-enhanced dye-penetration. LR lysate also attenuated the destruction of desmosomes after SLS treatment. Collectively, we demonstrated that LR lysate has protective effects on the skin barrier, which could expand the utility of probiotics to skin-moisturization ingredients.


Assuntos
Epiderme/efeitos dos fármacos , Lactobacillus rhamnosus/metabolismo , Modelos Biológicos , Probióticos/farmacologia , Administração Tópica , Anticorpos/farmacologia , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Desmossomos/efeitos dos fármacos , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Epiderme/patologia , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Irritantes/toxicidade , Proteínas de Membrana/metabolismo , Permeabilidade , Rodaminas/metabolismo , Proteínas de Junções Íntimas/metabolismo
5.
J Oleo Sci ; 68(8): 793-802, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292344

RESUMO

Atopic dermatitis (AD) is a cutaneous condition characterized by itchy, swollen, and dry skin, which is mediated by T helper cell-related cytokines. ß-Carotene, a natural red pigment found in plants, exhibits antioxidant activity that has been shown to promote an inflammatory response. Because it is not clear whether ß-carotene suppresses inflammation in AD skin tissues, we examined the effects of oral administration of ß-carotene in mice induced by a low zinc/magnesium diet (HR-AD diet). Our studies found that AD-like inflammation was remarkably reduced by ß-carotene. In addition, ß-carotene significantly suppressed protein expression of TNF-α, IL-1ß, and MCP-1 and mRNA expression of TSLP, IL-6, IL-1ß, IL-4, IL-5, and Par-2 in AD-like skin tissues. It was also found that mRNA and protein expression of filaggrin (a major structural protein in epidermis) in AD-like skin was significantly elevated by ß-carotene administration. Furthermore, ß-carotene treatment significantly reduced the activity and/or mRNA expression of matrix metalloproteinases (MMPs), degradation of the extracellular matrix and regulation of chemokines. These results suggest that ß-carotene reduces skin inflammation through the suppressed expression of inflammatory factors or the activity of MMPs as well as the promotion of filaggrin expression in AD-like skin. ß-Carotene is a potent anti-inflammatory agent, which improves AD-like skin by enhancing the skin barrier function.


Assuntos
Dermatite Atópica/tratamento farmacológico , beta Caroteno/uso terapêutico , Administração Oral , Animais , Citocinas/metabolismo , Dermatite Atópica/patologia , Dieta/efeitos adversos , Epiderme/patologia , Matriz Extracelular/metabolismo , Expressão Gênica/genética , Inflamação/tratamento farmacológico , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Camundongos Pelados , beta Caroteno/administração & dosagem
6.
PLoS Biol ; 17(7): e3000390, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31323028

RESUMO

Processes of molecular innovation require tinkering and shifting in the function of existing genes. How this occurs in terms of molecular evolution at long evolutionary scales remains poorly understood. Here, we analyse the natural history of a vast group of membrane-associated molecular systems in Bacteria and Archaea-the type IV filament (TFF) superfamily-that diversified in systems involved in flagellar or twitching motility, adhesion, protein secretion, and DNA uptake. The phylogeny of the thousands of detected systems suggests they may have been present in the last universal common ancestor. From there, two lineages-a bacterial and an archaeal-diversified by multiple gene duplications, gene fissions and deletions, and accretion of novel components. Surprisingly, we find that the 'tight adherence' (Tad) systems originated from the interkingdom transfer from Archaea to Bacteria of a system resembling the 'EppA-dependent' (Epd) pilus and were associated with the acquisition of a secretin. The phylogeny and content of ancestral systems suggest that initial bacterial pili were engaged in cell motility and/or DNA uptake. In contrast, specialised protein secretion systems arose several times independently and much later in natural history. The functional diversification of the TFF superfamily was accompanied by genetic rearrangements with implications for genetic regulation and horizontal gene transfer: systems encoded in fewer loci were more frequently exchanged between taxa. This may have contributed to their rapid evolution and spread across Bacteria and Archaea. Hence, the evolutionary history of the superfamily reveals an impressive catalogue of molecular evolution mechanisms that resulted in remarkable functional innovation and specialisation from a relatively small set of components.


Assuntos
Citoesqueleto/genética , DNA/metabolismo , Transferência Genética Horizontal/genética , Proteínas de Filamentos Intermediários/genética , Filamentos Intermediários/genética , Archaea/classificação , Archaea/genética , Archaea/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Adesão Celular/genética , Citoesqueleto/metabolismo , DNA/genética , Evolução Molecular , Proteínas de Filamentos Intermediários/classificação , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/classificação , Filamentos Intermediários/metabolismo , Movimento , Filogenia , Transporte Proteico/genética
7.
Nat Commun ; 10(1): 3056, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296869

RESUMO

Lamin A is a nuclear intermediate filament protein critical for nuclear architecture and mechanics and mutated in a wide range of human diseases. Yet little is known about the molecular architecture of lamins and mechanisms of their assembly. Here we use SILAC cross-linking mass spectrometry to determine interactions within lamin dimers and between dimers in higher-order polymers. We find evidence for a compression mechanism where coiled coils in the lamin A rod can slide onto each other to contract rod length, likely driven by a wide range of electrostatic interactions with the flexible linkers between coiled coils. Similar interactions occur with unstructured regions flanking the rod domain during oligomeric assembly. Mutations linked to human disease block these interactions, suggesting that this spring-like contraction can explain in part the dynamic mechanical stretch and flexibility properties of the lamin polymer and other intermediate filament networks.


Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Lamina Tipo A/metabolismo , Matriz Nuclear/metabolismo , Multimerização Proteica/fisiologia , Sequência de Aminoácidos/fisiologia , Animais , Cardiomiopatia Dilatada/genética , Reagentes para Ligações Cruzadas/química , Elasticidade , Humanos , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/isolamento & purificação , Lamina Tipo A/química , Lamina Tipo A/genética , Lamina Tipo A/isolamento & purificação , Espectrometria de Massas/métodos , Distrofias Musculares/genética , Mutação , Membrana Nuclear/metabolismo , Domínios Proteicos/genética , Estrutura Secundária de Proteína/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
8.
Iran J Immunol ; 16(2): 97-107, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31182684

RESUMO

Atopic dermatitis (AD) is characterized by skin inflammation, barrier dysfunction and chronic pruritus. In this review, recent advances in the pathogenesis of AD are summarized. Clinical efficacy of the anti-IL-4 receptor antibody dupilumab implies that type 2 cytokines IL-4 and IL-13 have pivotal roles in atopic inflammation. The expression of IL-4 and IL-13 as well as type 2 chemokines such as CCL17, CCL22 and CCL26 is increased in the lesional skin of AD. In addition, IL-4 and IL-13 down-regulate the expression of filaggrin in keratinocytes and exacerbate epidermal barrier dysfunction. Keratinocytes in barrier-disrupted epidermis produce large amounts of thymic stromal lymphopoietin, IL-25 and IL-33, conducing to type 2 immune deviation via OX40L/OX40 signaling. IL-31, produced by type 2 T cells, is a cardinal pruritogenic cytokine. IL-4 and IL-13 also amplify the IL-31-mediated sensory nerve signal. These molecules are particularly important targets for future drug development for AD.


Assuntos
Dermatite Atópica/imunologia , Epiderme/patologia , Queratinócitos/fisiologia , Células Th2/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Citocinas/metabolismo , Dermatite Atópica/tratamento farmacológico , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Ligante OX40/metabolismo , Receptores de Interleucina-4/imunologia , Receptores OX40/metabolismo , Transdução de Sinais
9.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31243049

RESUMO

Despite low-sequence homology, the intermediate filament (IF)-like protein FilP from Streptomyces coelicolor displays structural and biochemical similarities to the metazoan nuclear IF lamin. FilP, like IF proteins, is composed of central coiled-coil domains interrupted by short linkers and flanked by head and tail domains. FilP polymerizes into repetitive filament bundles with paracrystalline properties. However, the cations Na+ and K+ are found to induce the formation of a FilP hexagonal meshwork with the same 60-nm repetitive unit as the filaments. Studies of polymerization kinetics, in combination with EM techniques, enabled visualization of the basic building block-a transiently soluble rod-shaped FilP molecule-and its assembly into protofilaments and filament bundles. Cryoelectron tomography provided a 3D view of the FilP bundle structure and an original assembly model of an IF-like protein of prokaryotic origin, thereby enabling a comparison with the assembly of metazoan IF.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Bactérias/química , Biomarcadores , Cátions/química , Proteínas do Citoesqueleto/química , Imunofluorescência , Hifas , Proteínas de Filamentos Intermediários/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/ultraestrutura
10.
Biochem Med (Zagreb) ; 29(2): 020501, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31223255

RESUMO

There is an increasing number of experimental, genetic and clinical evidence of atopic dermatitis expression as a pre-condition for later development of other atopic diseases such as asthma, food allergy and allergic rhinitis. Atopic dermatitis is a heterogeneous, recurrent childhood disease, also present in the adult age. It is increasingly attributed to systemic features and is characterized by immunological and skin barrier integrity and function dysregulation. To maintain the protective function of the skin barrier, in particular the maintenance of pH, hydration and antimicrobial functions, the filaggrin, among others, plays a significant role. Filaggrin is a multifunctional, histidine-rich, insoluble protein. The lack of filaggrin is associated with various cutaneous (e.g. ichthyosis vulgaris, allergic contact dermatitis) and non-cutaneous (e.g. diabetes, inflammatory conditions of the gastrointestinal tract) diseases and may be a result of genetic, immunological factors combined with environmental factors. In this review we summarised (emphasized) recent findings in understanding the role of filaggrin in atopic dermatitis and other diseases, participants in the atopic march.


Assuntos
Dermatite Atópica , Proteínas de Filamentos Intermediários , Dermatite Atópica/diagnóstico , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Humanos , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo
11.
Nutrients ; 11(6)2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216667

RESUMO

With a complex etiology involving multiple factors, the condition known as itch is a primary symptom of many skin diseases. Current treatment methods are ineffective for addressing itches caused by dry skin, for example. We developed a botanical extract, ACTPER, made from a mixture of Actinidia arguta and Perilla frutescens, which have traditionally been used to treat itch. The quality of ACTPER as a research agent was controlled in our experiment by cell-based bioassays, as well as by high-performance liquid chromatography (HPLC), using two chemical markers. In the acetone-induced dry skin mice model, the oral administration of ACTPER alleviated dry skin-related skin properties and itching behavior. The RNA and protein expression of the filament aggregating protein (filaggrin) gene, a key factor involved in the regulation of skin barrier function, was significantly increased, as measured by quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay. To understand the underlying mechanism(s) at the molecular level, HaCaT cells, a human keratinocyte-derived cell line, were treated with various concentrations of ACTPER. We found that the protein expression of filaggrin was indeed upregulated by ACTPER in a dose dependent manner. Data from experiments involving the reporter plasmid containing the xenobiotic response element (XRE), and the chemical antagonist for the aryl hydrocarbon receptor (AhR), indicated that the ACTPER-mediated upregulation of filaggrin was controlled through the activation of the AhR signaling pathway. The molecular docking simulation study predicted that ACTPER might contain chemical compounds that bind directly to AhR. Taken together, our results suggest that ACTPER may provide the platform, based upon which a variety of safe and effective therapeutic agents can be developed to treat itch.


Assuntos
Actinidia/química , Proteínas de Filamentos Intermediários/metabolismo , Perilla frutescens/química , Extratos Vegetais/farmacologia , Prurido/tratamento farmacológico , Animais , Linhagem Celular , Humanos , Queratinócitos , Camundongos , Simulação de Acoplamento Molecular , Prurido/metabolismo , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Pele/metabolismo , Regulação para Cima/efeitos dos fármacos , Água
12.
Cells ; 8(5)2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31121896

RESUMO

Atopic dermatitis (AD) is characterized by dry and itchy skin evolving into disseminated skin lesions. AD is believed to result from a primary acquired or a genetically-induced epidermal barrier defect leading to immune hyper-responsiveness. Filaggrin (FLG) is a protein found in the cornified envelope of fully differentiated keratinocytes, referred to as corneocytes. Although FLG null mutations are strongly associated with AD, they are not sufficient to induce the disease. Moreover, most patients with ichthyosis vulgaris (IV), a monogenetic skin disease characterized by FLG homozygous, heterozygous, or compound heterozygous null mutations, display non-inflamed dry and scaly skin. Thus, all causes of epidermal barrier impairment in AD have not yet been identified, including those leading to the Th2-predominant inflammation observed in AD. Three dimensional organotypic cultures have emerged as valuable tools in skin research, replacing animal experimentation in many cases and precluding the need for repeated patient biopsies. Here, we review the results on IV and AD obtained with epidermal or skin equivalents and consider these findings in the context of human in vivo data. Further research utilizing complex models including immune cells and cutaneous innervation will enable finer dissection of the pathogenesis of AD and deepen our knowledge of epidermal biology.


Assuntos
Dermatite Atópica/patologia , Ictiose Vulgar/patologia , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Células Cultivadas , Dermatite Atópica/metabolismo , Humanos , Hipersensibilidade/metabolismo , Ictiose Vulgar/metabolismo , Inflamação/metabolismo , Modelos Biológicos , Mutação , Pele/metabolismo , Pele/patologia
13.
EMBO J ; 38(11)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31036554

RESUMO

To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 Å resolution. It revealed biochemical determinants for the A11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227K1, Y230K1, F231K1, and F234K1), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314K1 and L318K1). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314AK1, but not L318AK1, abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 Å structure of K1/10-1B containing a S233LK1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1S233L/K10-1B in solution without affecting secondary structure. The K1S233L/K10-1B octamer structure revealed S233LK1 causes aberrant hydrophobic interactions between 1B tetramers.


Assuntos
Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/metabolismo , Queratina-10 , Queratina-1 , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica/fisiologia , Substituição de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Citoesqueleto/química , Citoesqueleto/metabolismo , Difusão Dinâmica da Luz , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Filamentos Intermediários/genética , Queratina-1/química , Queratina-1/genética , Queratina-1/metabolismo , Queratina-10/química , Queratina-10/genética , Queratina-10/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologia
14.
PLoS One ; 14(4): e0214758, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973903

RESUMO

Myo/Nog cells are identified by their expression of the skeletal muscle specific transcription factor MyoD and the bone morphogenetic protein inhibitor noggin, and binding of the G8 monoclonal antibody. Their release of noggin is critical for morphogenesis and skeletal myogenesis. In the adult, Myo/Nog cells are present in normal tissues, wounds and skin tumors. Myo/Nog cells in the lens give rise to myofibroblasts that synthesize skeletal muscle proteins. The purpose of this study was to screen human lens tissue, rhabdomyosarcoma cell lines, and tissue sections from rhabdomyosarcoma, Wilms and tumors lacking features of skeletal muscle for co-localization of antibodies to Myo/Nog cell markers and the lens beaded filament proteins filensin and CP49. Immunofluorescence localization experiments revealed that Myo/Nog cells of the lens bind antibodies to beaded filament proteins. Co-localization of antibodies to G8, noggin, filensin and CP49 was observed in most RC13 and a subpopulation of RD human rhabdomyosarcoma cell lines. Western blotting with beaded filament antibodies revealed bands of similar molecular weights in RC13 and murine lens cells. Human alveolar, embryonal, pleomorphic and spindle cell rhabdomyosarcomas and Wilms tumors contained a subpopulation of cells immunoreactive for G8, noggin, MyoD and beaded filaments. G8 was also co-localized with filensin mRNA. Staining for beaded filament proteins was not detected in G8 positive cells in leiomyosarcomas, squamous and basal cell carcinomas, syringocarciomas and malignant melanomas. Lens beaded filament proteins were thought to be present only in the lens. Myo/Nog-like cells immunoreactive for beaded filaments may be diagnostic of tumors related to the skeletal muscle lineage.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteína MyoD/metabolismo , Rabdomiossarcoma/patologia , Tumor de Wilms/patologia , Animais , Anticorpos Monoclonais/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/imunologia , Cristalino/citologia , Cristalino/metabolismo , Camundongos , Microscopia de Fluorescência , Proteína MyoD/imunologia , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma Embrionário/metabolismo , Rabdomiossarcoma Embrionário/patologia , Tumor de Wilms/metabolismo
15.
Sci Transl Med ; 11(480)2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30787169

RESUMO

Skin barrier dysfunction has been reported in both atopic dermatitis (AD) and food allergy (FA). However, only one-third of patients with AD have FA. The purpose of this study was to use a minimally invasive skin tape strip sampling method and a multiomics approach to determine whether children with AD and FA (AD FA+) have stratum corneum (SC) abnormalities that distinguish them from AD without FA (AD FA-) and nonatopic (NA) controls. Transepidermal water loss was found to be increased in AD FA+. Filaggrin and the proportion of ω-hydroxy fatty acid sphingosine ceramide content in nonlesional skin of children with AD FA+ were substantially lower than in AD FA- and NA skin. These abnormalities correlated with morphologic changes in epidermal lamellar bilayer architecture responsible for barrier homeostasis. Shotgun metagenomic studies revealed that the nonlesional skin of AD FA+ had increased abundance of Staphylococcus aureus compared to NA. Increased expression of keratins 5, 14, and 16 indicative of hyperproliferative keratinocytes was observed in the SC of AD FA+. The skin transcriptome of AD FA+ had increased gene expression for dendritic cells and type 2 immune pathways. A network analysis revealed keratins 5, 14, and 16 were positively correlated with AD FA+, whereas filaggrin breakdown products were negatively correlated with AD FA+. These data suggest that the most superficial compartment of nonlesional skin in AD FA+ has unique properties associated with an immature skin barrier and type 2 immune activation.


Assuntos
Dermatite Atópica/diagnóstico , Hipersensibilidade Alimentar/diagnóstico , Pele/patologia , Adolescente , Área Sob a Curva , Criança , Pré-Escolar , Células Dendríticas/metabolismo , Dermatite Atópica/patologia , Epiderme/metabolismo , Hipersensibilidade Alimentar/patologia , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Lipídeos/análise , Microbiota , Pele/microbiologia , Fita Cirúrgica , Transcriptoma/genética , Perda Insensível de Água
16.
Exp Eye Res ; 185: 107585, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30790544

RESUMO

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations.


Assuntos
Aquaporinas/metabolismo , Água Corporal/metabolismo , Cálcio/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Processamento de Proteína Pós-Traducional , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Western Blotting , Caspases/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Cristalino/citologia , Células MCF-7/metabolismo , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Dados de Sequência Molecular , Miristatos/metabolismo , Oócitos , Domínios Proteicos , Transfecção , Xenopus laevis , Adulto Jovem
17.
Gene ; 692: 44-53, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30641223

RESUMO

Intermediate filaments (IF) belong to major cytoskeletal components of metazoan cells. We have previously determined a tissue specific expression and assembly properties of all eleven cytoplasmic IFs (IFA-1 - IFA-4, IFB-1, IFB-2, IFC-1, IFC-2, IFD-1, IFD-2, IFP-1) in C. elegans and reported an essential function for four (IFA-1, IFA-2, IFA-3 and IFB-1) of them. In this study we continued the characterisation of the IF proteins in C. elegans by searching for in vivo polymerisation partners of the IFA proteins. Using the murine IFA-1 to IFA-3-specific monoclonal Ab MH4 and the immunoprecipitation assay as a tool, we identified the heteropolymeric IFA-1/IFB-1 complexes in the whole nematode protein extract, confirming their existence also in vivo. Moreover, in the present study we also analysed evolutionary aspects of the IF proteins in C. elegans and in nematodes. We found 106 C. elegans IF homologs in different nematode clades. Phylogenetic analyses suggest that all nematode IFs (including the three newly identified IF sequences IFA-5, IFCDP-1 and IFCDP-2) might arose from a AB-type IF ancestor through repeated gene duplications and sequence divergence. Interestingly, the C. elegans IF proteins IFA-1 and IFB-1 represent a heteropolymeric IF cytoskeleton in all investigated nematode clades, in contrast to other sequences restricted to the clade III-V (IFA-2, IFA-4), III (IFA-5) and V (IFB-2, IFCDP) taxa, or even to the Caenorhabditis genus (IFA-3, IFC-1 to IFP-1). These analyses provide an insight into the origin of the multiple IFs in nematodes and also represent a basis for further studies of these sequences in nematodes.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Filamentos Intermediários/genética , Filogenia , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Evolução Molecular , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Complexos Multiproteicos , Nematoides/genética
18.
Skin Res Technol ; 25(4): 424-433, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30657212

RESUMO

OBJECTIVE: The objective of this study was to perform noninvasive analysis of skin proteins in a healthy Chinese population using label-free nanoflow liquid chromatography-mass spectrometry (nLC-MS). MATERIALS AND METHODS: Five consecutive tape strippings were obtained from the volar forearm skin of healthy Chinese subjects. Proteins were extracted, and trypsin-digested peptides were analyzed by a nanochromatography instrument coupled to an Orbitrap Fusion Tribrid mass spectrometer. Data-dependent acquisition allowed protein identification, which was performed by using Proteome Discoverer software (v2.2). RESULTS: In this study, we identified 80 common proteins that were expressed in the skin of healthy Chinese volunteers and divided these proteins into 16 categories, including keratins, cornified envelope proteins, and enzymes associated with substance metabolism. These proteins were closely associated with multiple functions of the skin barrier. CONCLUSION: This study provides a noninvasive method to analyze healthy human epidermal proteins, which are closely associated with the skin barrier. In addition, this study provides a reference for further studies on the application of proteomic technologies to investigate the role of human epidermal proteins in health and disease.


Assuntos
Espectrometria de Massas/instrumentação , Proteínas/metabolismo , Pele/metabolismo , Grupo com Ancestrais do Continente Asiático/etnologia , Epiderme/metabolismo , Voluntários Saudáveis , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Proteômica/métodos , Software
19.
J Cosmet Laser Ther ; 21(4): 217-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30198801

RESUMO

Background: Fractional carbon dioxide laser resurfacing (FxCR) is a routine treatment of Dermatology while many patients suffered the damage of skin barrier function after FxCR. Objective: To evaluate the benefits of antimicrobial peptides (AMPs) and hyaluronic acid (HA) compound mask on wound healing after FxCR on human and mouse skin. Methods: Twenty-four subjects were treated with FxCR on the bilateral cheeks. AMPs and HA compound mask was applied on the FxCR-treated area of left cheek. The erythema index (EI), melanin index (MI), transepidermal water loss (TEWL) of FxCR-treated areas on both cheeks were measured. By HE staining, immunohistostaing and western blotting, we analyzed epidermal thickness, FLG, IVL expression and protein levels of cramp in FxCR treated dorsal mice skin. Results: The EI, MI, and TEWL in the AMPs and HA compound mask-treated area of left cheek were significantly lower than those in the untreated area of right cheek. Topically application of AMPs and HA compound mask reduced thickening of mouse skin and also result in an increase in the production of FLG, IVL and cramp. Conclusion: Application of AMPs and HA compound mask is an effective method for enhancing wound healing after FxCR, by reducing transient adverse effects such as erythema, hyperpigmentation, and increased TEWL.


Assuntos
Anti-Infecciosos/uso terapêutico , Bochecha , Ácido Hialurônico/uso terapêutico , Lasers de Gás/uso terapêutico , Regeneração da Pele por Plasma/métodos , Cicatrização/efeitos dos fármacos , Adulto , Animais , Dióxido de Carbono , Eritema/etiologia , Feminino , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Melaninas/metabolismo , Camundongos , Perda Insensível de Água
20.
J Histochem Cytochem ; 67(2): 85-97, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30199656

RESUMO

Filaggrin (FLG) and corneodesmosin (CDSN) are two key proteins of the human epidermis. FLG loss-of-function mutations are the strongest genetic risk factors for human atopic dermatitis. Studies of the epidermal distribution of canine FLG and CDSN are limited. Our aim was to better characterize the distribution of FLG and CDSN in canine skin. Using immunohistochemistry on beagle skin, we screened a series of monoclonal antibodies (mAbs) specific for human FLG and CDSN. The cross-reactive mAbs were further used using immunoelectron microscopy and Western blotting. The structure of canine CDSN and FLG was determined using publicly available databases. In the epidermis, four anti-FLG mAbs stained keratohyalin granules in the granular keratinocytes and corneocyte matrix of the lower cornified layer. In urea-extracts of dog epidermis, several bands corresponding to proFLG and FLG monomers were detected. One anti-CDSN mAb stained the cytoplasm of granular keratinocytes and cells of both the inner root sheath and medulla of hair follicles. Dog CDSN was located in lamellar bodies, in the extracellular parts of desmosomes and in corneodesmosomes. A protein of 52 kDa was immunodetected. Genomic DNA analysis revealed that the amino acid sequence and structure of canine and human CDSN were highly similar.


Assuntos
Glicoproteínas/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Pele/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Cães , Regulação da Expressão Gênica , Glicoproteínas/química , Glicoproteínas/imunologia , Imunoquímica , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/imunologia , Transporte Proteico
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