RESUMEN
Silica aerogels are high-performance thermal insulation materials that can be used to provide unique solutions in the envelopes of buildings when space is limited. They are most often applied in historic buildings due to thin insulation thicknesses and since they are compatible with historic structures. In 2021, the first Aerogel Architecture Award was held at Empa in Switzerland in order to collect, evaluate and award outstanding uses of this relatively new building material. From the submitted projects, three were selected for an award by an expert jury. They showcased applications in which heritage protection and the conservation of a building's character and expression were reconciled with significant improvements in the energy efficiency of the building. The submissions also showed that a broader communication of these types of solutions is important in order to provide more information and security to planners and heritage offices and to facilitate the application of these materials in the future so that they can contribute to the protection of cultural heritage and reductions in the operational and embodied emissions of our building stock by extending the life expectancy and energy efficiency of existing buildings.
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Bird feather lipids are usually attributed to the oily secretion product of the uropygial (preen) gland. We have observed, however, that feathers exhibit a strong reaction with osmium tetroxide (OsO4), even after treatment with detergents. This leads us to postulate the existence of endogenous feather lipids distinct from preen gland lipids. In order to substantiate our hypothesis, we investigated down feathers from a 1-day-old chicken as their uropgygial gland is not functionally active. The results confirmed the osmiophilic reaction, which was concentrated in the center of barbs and strongly reduced after lipid extraction. In these lipid extracts, we identified using thin layer chromatography, cholesterol, various ceramides, glycolipids, phospholipids, and fatty acids, which closely resembled the lipid composition of the water barrier in the chicken-cornified epidermal envelope. This composition is clearly distinct from chicken uropygeal gland secretion (UGS) known to consist of fatty alcohols as part of aliphatic monoester waxes and of free, predominantly saturated, fatty acids. A filter assay showed a strong reactivity between OsO4 and the fatty acids C18:1 and C18:2 and with feather lipid extracts, but not with UGS. These observations were confirmed by gas chromatography detecting unsaturated fatty acids including C18:1 and C18:2 as well as cholesterol exclusively in chicken feathers. Our results indicate that (1) endogenous lipids are detectable in chicken feathers and distinct from UGS and (2) in analogy to the morphogenesis of the cornified envelope of chicken feather lipids that may have derived from cellular feather-precursors, apparently enduring the specific cell death during developmental feather cornification.
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Plumas/química , Lípidos/química , Glándulas Sebáceas/química , Animales , PollosRESUMEN
OBJECTIVE: Autoimmune hepatitis and primary sclerosing cholangitis are chronic inflammatory disorders of unknown aetiology, frequently associated with the presence of perinuclear antineutrophil cytoplasmic antibodies (p-ANCAs) directed against an unknown antigen of myeloid cells. METHODS AND RESULTS: Here, it is reported that p-ANCAs in autoimmune liver disorders react with beta-tubulin isotype 5 (TBB-5) as autoantigen as well as with its evolutionary bacterial precursor protein FtsZ. Both proteins were confirmed as antigens of p-ANCAs in autoimmune liver disorders by demonstrating reactivity of ANCA-positive sera with recombinant TBB-5 (72-88%) and FtsZ (64-82%) on immunoblots and antigen-specific abrogation of ANCA immunofluorescence when sera had been preabsorbed with tubulin and FtsZ. Using sera from interleukin 10-deficient mice (Il10(-)/(-)), an animal model of inflammatory bowel disease, it was also demonstrated that antibodies against TBB-5 are generated in response to intestinal microorganisms. However, unlike autoimmune liver disorders, human antibodies to FtsZ in the absence of TBB-5 antibodies were also a frequent finding in non-autoimmune liver diseases (up to 95%). Reactivity to TBB-5 without the presence of FtsZ antibodies was found in very few cases (<1%) in autoimmune liver disorders. CONCLUSIONS: Thus, p-ANCAs in autoimmune liver diseases are directed against human TBB-5 cross-reacting with the bacterial protein FtsZ, probably reflecting an abnormal immune response to intestinal microorganisms in susceptible, possibly genetically predisposed individuals.
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Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Proteínas Bacterianas/inmunología , Colangitis Esclerosante/inmunología , Proteínas del Citoesqueleto/inmunología , Hepatitis Autoinmune/inmunología , Tubulina (Proteína)/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Reacciones Antígeno-Anticuerpo/inmunología , Autoantígenos/análisis , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Humanos , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Mapeo Peptídico/métodos , Proteínas Recombinantes/inmunología , Adulto JovenRESUMEN
The thyroid gland has an exceptionally high selenium content, even during selenium deficiency. At least 11 selenoproteins are expressed, which may be involved in the protection of the gland against the high amounts of H2O2 produced during thyroid hormone biosynthesis. As determined here by in situ hybridization and Northern blotting experiments, glutathione peroxidases (GPx) 1 and 4 and selenoprotein P were moderately expressed, occurring selectively in the follicular cells and in leukocytes of germinal follicles of thyroids affected by Hashimoto's thyroiditis. Selenoprotein 15 was only marginally expressed and distributed over all cell types. GPx3 mRNA was exclusively localized to the thyrocytes, showed the highest expression levels and was down-regulated in 5 of 6 thyroid cancer samples as compared to matched normal controls. GPx3 could be extracted from thyroidal colloid by incubation with 0.5% sodium dodecyl sulfate indicating that this enzyme is (i) secreted into the follicular lumen and (ii) loosely attached to the colloidal thyroglobulin. These findings are consistent with a role of selenoproteins in the protection of the thyroid from possible damage by H2O2. Particularly, GPx3 might use excess H2O2 and catalyze the polymerization of thyroglobulin to the highly cross-linked storage form present in the colloid.
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Glutatión Peroxidasa/metabolismo , Selenoproteínas/metabolismo , Glándula Tiroides/enzimología , Northern Blotting , Línea Celular Tumoral , Glutatión Peroxidasa/análisis , Glutatión Peroxidasa/genética , Humanos , Peróxido de Hidrógeno/metabolismo , ARN Mensajero/metabolismo , Glándula Tiroides/metabolismoRESUMEN
During mammalian embryogenesis the emerging epidermis is temporarily covered by an epithelial monolayer, the periderm. In chicken, a second epithelial layer, the subperiderm, located underneath the periderm develops in later embryogenesis. Together the periderm and the subperiderm are referred to as the PSP unit. The cells of the PSP unit are tightly connected by tight junctions (TJ), thereby providing the embryo with an impermeable bilayered diffusion barrier. The emerging epidermis assumes its barrier function by cornification beginning at embryonic day 17 (E17) before at E18 the PSP unit undergoes desquamation. Lipid analysis of both epithelia after their mechanical separation revealed a dramatic increase to about 100-fold values of barrier-relevant ceramides, i.e. those known to essentially contribute to the diffusion barrier of the cornified envelope, in the emerging epidermis between E17 and E19. In contrast, the content of barrier-relevant ceramides in the PSP unit remained at constantly low levels throughout embryogenesis. These data strongly argue in favour of different mechanisms for the barrier function of the two epithelia. TJ in the PSP unit provide the main diffusion barrier protecting the embryo until beginning of desquamation at E18. At this developmental stage the content of cornified envelope-specific ceramides is substantially elevated, thus enabling the epidermis to fulfil its function as the major diffusion barrier after desquamation of the PSP unit. The observation that barrier-relevant ceramides are formed prior to desquamation of the PSP unit points to a precisely regulated sequence in that desquamation does not occur until the lipid-based barrier of the cornified envelope is completed and suggests in addition that these lipids might be essential regulators of the interaction between the PSP unit and the emerging epidermis.
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Ceramidas/metabolismo , Desarrollo Embrionario , Epidermis/embriología , Epidermis/metabolismo , Animales , Embrión de Pollo , Epidermis/ultraestructura , Regulación hacia ArribaRESUMEN
Laminin-5 is a major adhesion protein of the skin basement membrane and crucially involved in integrin-mediated cell substrate attachment of keratinocytes, which is important for hemidesmosomal anchorage as well as for keratinocyte migration during epidermal wound healing. To investigate its role in keratinocyte migration, we analyzed laminin-5-deficient cells of patients with a lethal variant of junctional epidermolysis bullosa. Normal migrating keratinocytes adopted monopolar morphology with a distinct front lamella and employed a continuous mode of translocation. In contrast, laminin-5-deficient cells assumed a stretched bipolar shape with two lamella regions and migrated in a discontinuous, saltatory manner characterized by significantly decreased directional persistence and reduced migration velocity. The distinct morphology as well as the migratory phenotype apparently resulted from a defect in the formation of cell substrate adhesions that were completely missing in the cell body and less stable in the lamella regions. Accordingly in normal keratinocytes, a bipolar shape and a saltatory migration mode were inducible by blocking laminin-5-mediated substrate adhesion. Our findings clearly point to an essential role of laminin-5 in forming dynamic cell substrate adhesion during migration of epidermal keratinocytes and provide an explanation for the cellular mechanisms that underlie the lethal form of junctional epidermolysis bullosa.
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Moléculas de Adhesión Celular/fisiología , Movimiento Celular , Queratinocitos/fisiología , Adhesión Celular , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Polaridad Celular , Forma de la Célula , Células Cultivadas , Epidermólisis Ampollosa de la Unión/metabolismo , Epidermólisis Ampollosa de la Unión/patología , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Seudópodos/fisiología , KalininaRESUMEN
To address the functions of Rac1 in keratinocytes of the basal epidermal layer and in the outer root sheath of hair follicles, we generated transgenic mice expressing a dominant inhibitory mutant of Rac, N17Rac1, under the control of the keratin 14 promoter. These mice do not exhibit an overt skin phenotype but show protracted skin wound re-epithelialization. Investigation into the underlying mechanisms revealed that in vivo both proliferation of wound-edge keratinocytes and centripetal migration of the neo-epidermis were impaired. Similar results were obtained in mice with an epidermis-specific deletion of Rac1. Primary epidermal keratinocytes that expressed the N17Rac1 transgene were less proliferative than control cells and showed reduced ERK1/2 phosphorylation upon growth factor stimulation. Adhesion, spreading, random migration and closure of scratch wounds in vitro were significantly inhibited on collagen I and, to a lesser extent, on fibronectin. Stroboscopic analysis of cell dynamics (SACED) of N17Rac1 transgenic and control keratinocytes identified decreased lamella-protrusion persistence in connection with increased ruffle frequency as a probable mechanism for the observed impairment of keratinocyte adhesion and migration. We conclude that Rac1 is functionally required for normal epidermal wound healing and, in this context, exerts a dual function - namely the regulation of keratinocyte proliferation and migration.
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Epidermis/fisiopatología , Cicatrización de Heridas , Proteína de Unión al GTP rac1/fisiología , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular , Queratinocitos/citología , Proteína de Unión al GTP rac1/genéticaRESUMEN
For the invasive migration of tumor cells, at least two mechanisms are currently discussed: (1) the mesenchymal mode depending on extracellular proteolysis and (2) the proteolysis-independent amoeboid mode depending on the activity of the Rho kinase ROCK. The ability of tumor cells to switch between different modes of motility has been shown to limit the efficiency of agents aimed to reduce invasion. Here we show by combining 2D and 3D migration assays that human mammary tumor cells exhibited a strongly reduced migration velocity as compared to their normal counterparts indicating that high invasiveness is not necessarily correlated with high migratory capacity in 2D assays. This reduced migration was apparently due to significant differences in actin organization, decreased persistence of lamellipodia by 50% and increased cell substrate adhesion. These differences resulted from a 2.5-fold higher activity of ROCK and were mediated by its downstream effectors myosin light chain kinase and cofilin. Thus, inhibition of ROCK activity caused a marked increase in 2D migration efficiency by 40%, without, however, affecting 3D invasion. A massive reduction of invasion by 60% was achieved by the simultaneous inhibition of the ROCK-dependent amoeboid and the extracellular proteolysis-dependent mesenchymal mode. These results may point to a new efficient strategy for blocking tumor cell invasion in vivo.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Línea Celular Tumoral , Movimiento Celular/fisiología , Femenino , Adhesiones Focales , Humanos , Técnicas In Vitro , Cadenas Ligeras de Miosina/metabolismo , Invasividad Neoplásica/fisiopatología , Fenotipo , Fosforilación , Seudópodos/fisiología , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rhoRESUMEN
Growing evidence shows that the soluble N-terminal form (sAPPalpha) of the amyloid precursor protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation, migration and adhesion. APP is a member of a protein family including the two mammalian amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro, which could be completely rescued by either exogenously added recombinant sAPPalpha, or by co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-knockout keratinocytes revealed reduced migration velocity resulting from severely compromised cell substrate adhesion. Keratinocytes from double knockout mice died within the first week of culture, indicating essential functions of APP-family members for survival in vitro. Our data indicate that sAPPalpha has to be considered as an essential epidermal growth factor which, however, in vivo can be functionally compensated to a certain extent by other growth factors, e.g., factors released from dermal fibroblasts.
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Precursor de Proteína beta-Amiloide/fisiología , Movimiento Celular , Proliferación Celular , Queratinocitos/citología , Queratinocitos/fisiología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/farmacología , Animales , Adhesión Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Queratinocitos/efectos de los fármacos , Ratones , Ratones Noqueados , Microscopía por Video , Proteínas Recombinantes/genéticaRESUMEN
Dendritic cells (DC) change their phenotype and functional properties during maturation. CD83 cell surface expression is induced on mature DC (mDC). In this study, we investigated intracellular CD83 localization and transport in human monocyte-derived DC. The enhanced level of CD83 cell surface expression in mDC resulted predominantly from increased protein synthesis, and in addition from regulated intracellular transport of CD83 protein. An internal pool of CD83 protein is present in immature DC (iDC). Although CD83 protein in iDC and in mDC was localized in the Golgi compartment and in recycling endosomes, only in mature cells did CD83 co-localize with MHC class II molecules in endocytic vesicles. CD83 cell surface expression on iDC was induced by inhibition of endocytosis. This result could be explained by CD83 cycling between endosomes and the cell surface in iDC. The mDC also rapidly internalized membrane-bound CD83 protein. Furthermore, a thiol protease inhibitor and specific cathepsin inhibitors impaired CD83 up-regulation in DC, indicating a role of endosomal proteases in the maturation-induced exposure of CD83 on the plasma membrane.
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Células Dendríticas/inmunología , Inmunoglobulinas/inmunología , Glicoproteínas de Membrana/inmunología , Antígenos CD , Endosomas/inmunología , Aparato de Golgi/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Factores de Tiempo , Antígeno CD83RESUMEN
Misfolded proteins are removed from the ER (endoplasmic reticulum) by retrotranslocation to the cytosol and degradation by the ubiquitin-proteasome system in a process designated ERAD (ER-associated degradation). Analysing the turnover of a misfolded form of the ER-resident chaperone BiP (heavy-chain binding protein) (BiPDeltaA), we found that the degradation of BiPDeltaA did not follow this general ERAD pathway. In transfected cells, BiPDeltaA was degraded, although proteasome-dependent ERAD was inactivated either by proteasome inhibitors or by ATP depletion. In semi-permeabilized cells, which did not support the degradation of the proteasomal substrate alpha1-antitrypsin, the degradation of BiPDeltaA was still functional, excluding the Golgi apparatus or lysosomes as the degradative compartment. The degradation of BiPDeltaA was recapitulated in biosynthetically loaded brain microsomes and in an extract of luminal ER proteins. In contrast with proteasome-dependent ERAD, degradation fragments were detectable inside the microsomes and in the extract, and the degradation was prevented by a serine protease inhibitor. These results show that the degradation of BiPDeltaA was initiated in the ER lumen by a serine protease, and support the view that proteasome-independent ERAD pathways exist.
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Proteínas Portadoras/metabolismo , Retículo Endoplásmico/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , Animales , Células CHO , Cricetinae , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Pliegue de Proteína , Transporte de Proteínas , Serina Endopeptidasas/metabolismoRESUMEN
During epithelial cell migration, membrane ruffles can be visualized by phase contrast microscopy as dark waves arising at the leading edge of lamellipodia that move centripetally toward the main cell body. Despite the common use of the term membrane ruffles, their structure, molecular composition, and the mechanisms leading to their formation remained largely unknown. We show here that membrane ruffles differ from the underlying cell lamella by more densely packed bundles of actin filaments that are enriched in the actin cross-linkers filamin and ezrin, pointing to a specific bundling process based on these cross-linkers. The accumulation of phosphorylated, that is, inactivated, cofilin in membrane ruffles suggests that they are compartments of inhibited actin filament turnover. High Rac1 and low RhoA activities were found under conditions of suboptimal integrin-ligand interaction correlating with low lamellipodia persistence, inefficient migration, and high ruffling rates. Based on these findings, we define membrane ruffles as distinct compartments of specific composition that form as a consequence of inefficient lamellipodia adhesion.
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Citoesqueleto de Actina/metabolismo , Estructuras de la Membrana Celular/metabolismo , Movimiento Celular/fisiología , Células Epiteliales/metabolismo , Seudópodos/metabolismo , Citoesqueleto de Actina/ultraestructura , Factores Despolimerizantes de la Actina , Estructuras de la Membrana Celular/ultraestructura , Células Cultivadas , Proteínas Contráctiles/metabolismo , Proteínas del Citoesqueleto , Células Epiteliales/ultraestructura , Fibronectinas/metabolismo , Filaminas , Humanos , Integrina alfa5beta1/metabolismo , Queratinocitos/metabolismo , Queratinocitos/ultraestructura , Ligandos , Masculino , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica de Rastreo , Fosfoproteínas/metabolismo , Seudópodos/ultraestructura , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier.
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Apoptosis , Epidermis/crecimiento & desarrollo , Piel/embriología , Piel/patología , Uniones Estrechas/química , Animales , Caspasas/metabolismo , Pollos , Claudina-1 , Desarrollo Embrionario y Fetal , Células Epidérmicas , Epidermis/química , Peroxidasa de Rábano Silvestre/metabolismo , Etiquetado Corte-Fin in Situ , Proteínas de la Membrana/metabolismo , Ocludina , Uniones Estrechas/ultraestructuraRESUMEN
The soluble form of the beta-amyloid precursor protein (sAPPalpha) is known to function in the autocrine regulation of epidermal growth and repair. Here we show that its proteolytic release by alpha-secretase in normal human keratinocytes is susceptible to hydroxamic-acid-based zinc metalloproteinase inhibitors and suppressed by these inhibitors by 80%-90%. As various other growth factors participate in regulating epidermal growth we investigated whether the inhibitor-induced sAPPalpha-deficiency would affect keratinocyte proliferation. At optimal inhibitor concentrations the suppression of sAPPalpha-release was followed by a decline in proliferation by 50%-60%, indicating that sAPPalpha is a major growth factor that cannot be compensated for by other growth factors. This finding was the basis for the treatment of human lesional psoriatic keratinocytes with these inhibitors, which resulted in the normalization of their increased proliferation rates. The reversibility of these effects and the lack of toxicity underline the value of these inhibitors and suggest their therapeutic application in psoriatic skin diseases.
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Precursor de Proteína beta-Amiloide/metabolismo , Queratinocitos/metabolismo , Queratinocitos/patología , Psoriasis/metabolismo , Psoriasis/patología , Adulto , Anciano , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Biopsia , División Celular , Células Cultivadas , Endopeptidasas/metabolismo , Humanos , Persona de Mediana Edad , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
More than 97% of mice in which the C-terminal region of connexin43 (Cx43) was removed (designated as Cx43K258stop) die shortly after birth due to a defect of the epidermal barrier. The abnormal expression of Cx43K258stop protein in the uppermost layers of the epidermis seems to perturb terminal differentiation of keratinocytes. In contrast to Cx43-deficient mice, neonatal Cx43K258stop hearts show no lethal obstruction of the right ventricular outflow tract, but signs of dilatation. Electrocardiographies of neonatal hearts reveal repolarization abnormalities in 20% of homozygous Cx43K258stop animals. The very rare adult Cx43K258stop mice show a compensation of the epidermal barrier defect but persisting impairment of cardiac function in echocardiography. Female Cx43K258stop mice are infertile due to impaired folliculogenesis. Our results indicate that the C-terminally truncated Cx43K258stop mice lack essential functions of Cx43, although the truncated Cx43 protein can form open gap junctional channels.
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Conexina 43/metabolismo , Epidermis/anomalías , Epidermis/metabolismo , Animales , Animales Recién Nacidos , Biomarcadores , Diferenciación Celular , Conexina 43/química , Conexina 43/genética , Electrocardiografía , Epidermis/química , Femenino , Proteínas Filagrina , Uniones Comunicantes/metabolismo , Corazón/fisiología , Cardiopatías Congénitas , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/citología , Miocardio/metabolismo , Ovario/anomalías , Ovario/metabolismo , Fosfoproteínas/metabolismo , Tasa de Supervivencia , Proteína de la Zonula Occludens-1RESUMEN
The paradigm of endoplasmic reticulum (ER)-associated degradation (ERAD) holds that misfolded secretory and membrane proteins are translocated back to the cytosol and degraded by the proteasome in a coupled process. Analyzing the degradation of ER-localized amyloid beta-peptide (Abeta), we found a divergence from this general model. Cell-free reconstitution of the export in biosynthetically loaded ER-derived brain microsomes showed that the export was mediated by the Sec61p complex and required a cytosolic factor but was independent of ATP. In contrast to the ERAD substrates known so far, the exported Abeta was degraded by both, a proteasome-dependent and a proteasome-independent pathway. RNA interference experiments in Abeta-transfected cells identified the protease of the proteasome-independent pathway as insulin-degrading enzyme (IDE). The IDE-mediated clearance mechanism for ER-localized Abeta represents an as yet unknown type of ERAD which is not entirely dependent on the proteasome.
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Péptidos beta-Amiloides/metabolismo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Fragmentos de Péptidos/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/citología , Encéfalo/metabolismo , Células CHO , Cricetinae , Cisteína Endopeptidasas/metabolismo , Células HeLa , Humanos , Insulisina/metabolismo , Proteínas de la Membrana/metabolismo , Microsomas/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Complejo de la Endopetidasa Proteasomal , Señales de Clasificación de Proteína , Transporte de Proteínas/fisiología , Interferencia de ARN , Canales de Translocación SEC , PorcinosRESUMEN
The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.
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Microdominios de Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ácidos Mirísticos/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Ácido Palmítico/metabolismo , Seudópodos/ultraestructura , Acilación , Animales , Células CHO , Línea Celular , Cricetinae , Células Epiteliales/ultraestructura , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Ratas , TransfecciónRESUMEN
Quality control mechanisms in the endoplasmic reticulum (ER) ensure that misfolded proteins are recognized and targeted for degradation. According to the current view of ER-associated degradation (ERAD), the degradation does not occur in the ER itself but requires the retrotranslocation of the proteins to the cytosol where they are degraded by proteasomes. Although this model appears to be valid for many different proteins a number of exceptions from this rule suggest that additional proteasome-independent ERAD pathways may exist. In this review, we will summarize what is known about these alternative ERAD pathways.
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Retículo Endoplásmico/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de ProteínaRESUMEN
The amyloid precursor protein (APP) was initially detected in cells of the central nervous system where it is considered to be involved in the pathogenesis of Alzheimer's disease. However, APP is also found in peripheral organs with exceptionally strong expression in the mammalian epidermis where it fulfils a variety of distinct biological roles. Full length APP appears to facilitate keratinocyte adhesion due to its ability to interact with the extracellular matrix. The C-terminus of APP also serves as adapter protein for binding the motor protein kinesin thereby mediating the centripetal transport of melanosomes in epidermal melanocytes. By the action of alpha-secretase sAPPalpha, the soluble N-terminal portion of APP, is released. sAPPalpha has been shown to be a potent epidermal growth factor thus stimulating proliferation and migration of keratinocytes as well as the exocytic release of melanin by melanocytes. The release of sAPPalpha can be almost completely blocked by inhibiting alpha-secretase with hydroxamic acid-based zinc metalloproteinase inhibitors. In hyperproliferative keratinocytes from psoriatic skin this inhibition results in normalized growth.
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Precursor de Proteína beta-Amiloide/fisiología , Epidermis/metabolismo , Queratinocitos/fisiología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/fisiología , Humanos , Queratinocitos/metabolismoRESUMEN
sAPPalpha, the soluble form of the beta-amyloid precursor protein, has been shown to act as a potent epidermal growth factor by stimulating keratinocyte proliferation and migration. In this report we provide evidence for a cytoprotective role of sAPPalpha. As a model we used HaCaT cells and normal human keratinocytes (NHK) cultured in the absence of fetal calf serum and bovine pituitary extract. Under these conditions keratinocytes began to undergo apoptosis at increasing rates after 96 h of culture. Surprisingly, keratinocytes were protected from apoptosis by the addition of 50 nM recombinant sAPPalpha. Subsequent experiments were performed to elucidate the regulatory basis of the cytoprotective role of sAPPalpha. We found that recombinant sAPPalpha facilitated the substrate adhesion of keratinocytes in the first 30 minutes after seeding. The basis for this adhesion-promoting function was shown by the ability of recombinant sAPPalpha to continuously coat the culture dish thereby promoting the ability to bind keratinocytes. A second mechanism explaining the cytoprotective role was found in the significant inhibition of apoptosis by recombinant sAPPalpha. In HaCaT cells moderate UV-B irradiation was sufficient to induce apoptosis. In contrast, induction of apoptosis in NHK required additionally the depletion of endogenous sAPPalpha suggesting that sAPPalpha mediates protection against UV-B irradiation. Staurosporine-induced apoptosis rates were significantly reduced by about 59% after addition of recombinant sAPPalpha. These results show that sAPPalpha exerts a pronounced cytoprotective effect and that this effect is mediated by facilitated cell adhesion and by the antiapoptotic function of sAPPalpha.