RESUMEN
OBJECTIVE: Even though genetic predisposition has proven to be an important element in Parkinson's disease (PD) etiology, monozygotic (MZ) twins with PD displayed a concordance rate of only about 20% despite their shared identical genetic background. METHODS: We recruited 5 pairs of MZ twins discordant for idiopathic PD and established skin fibroblast cultures to investigate mitochondrial phenotypes in these cellular models against the background of a presumably identical genome. To test for genetic differences, we performed whole genome sequencing, deep mitochondrial DNA (mtDNA) sequencing, and tested for mitochondrial deletions by multiplex real-time polymerase chain reaction (PCR) in the fibroblast cultures. Further, the fibroblast cultures were tested for mitochondrial integrity by immunocytochemistry, immunoblotting, flow cytometry, and real-time PCR to quantify gene expression. RESULTS: Genome sequencing did not identify any genetic difference. We found decreased mitochondrial functionality with reduced cellular adenosine triphosphate (ATP) levels, altered mitochondrial morphology, elevated protein levels of superoxide dismutase 2 (SOD2), and increased levels of peroxisome proliferator-activated receptor-gamma coactivator-α (PPARGC1A) messenger RNA (mRNA) in skin fibroblast cultures from the affected compared to the unaffected twins. Further, there was a tendency for a higher number of somatic mtDNA variants among the affected twins. INTERPRETATION: We demonstrate disease-related differences in mitochondrial integrity in the genetically identical twins. Of note, the clinical expression matches functional alterations of the mitochondria. ANN NEUROL 2021;89:158-164.
Asunto(s)
ADN Mitocondrial/genética , Predisposición Genética a la Enfermedad/genética , Mitocondrias/genética , Enfermedad de Parkinson/metabolismo , Gemelos Monocigóticos/genética , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Enfermedad de Parkinson/genética , FenotipoRESUMEN
Allergic asthma is a chronical pulmonary disease with high prevalence. It manifests as a maladaptive immune response to common airborne allergens and is characterized by airway hyperresponsiveness, eosinophilia, type 2 cytokine-associated inflammation, and mucus overproduction. Alveolar macrophages (AMs), although contributing to lung homeostasis and tolerance to allergens at steady state, have attracted less attention compared to professional antigen-presenting and adaptive immune cells in their contributions. Using an acute model of house dust mite-driven allergic asthma in mice, we showed that a fraction of resident tissue-associated AMs, while polarizing to the alternatively activated M2 phenotype, exhibited signs of polynucleation and polyploidy. Mechanistically, in vitro assays showed that only Granulocyte-Macrophage Colony Stimulating Factor and interleukins IL-13 and IL-33, but not IL-4 or IL-5, participate in the establishment of this phenotype, which resulted from division defects and not cell-cell fusion as shown by microscopy. Intriguingly, mRNA analysis of AMs isolated from allergic asthmatic lungs failed to show changes in the expression of genes involved in DNA damage control except for MafB. Altogether, our data support the idea that upon allergic inflammation, AMs undergo DNA damage-induced stresses, which may provide new unconventional therapeutical approaches to treat allergic asthma.
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Asma/etiología , Asma/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-33/farmacología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Poliploidía , Animales , Asma/patología , Biomarcadores , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Técnica del Anticuerpo Fluorescente , Expresión Génica , Células Gigantes/efectos de los fármacos , Células Gigantes/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Activación de Macrófagos , Macrófagos Alveolares/citología , RatonesRESUMEN
Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1- but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1- cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.
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Alérgenos/inmunología , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/metabolismo , Células Dendríticas/inmunología , Tolerancia Inmunológica , Pulmón/inmunología , Receptor de Anafilatoxina C5a/metabolismo , Animales , Antígeno CD11b/inmunología , Antígenos CD40/inmunología , Diferenciación Celular/inmunología , Proliferación Celular/genética , Técnicas de Cocultivo , Complemento C5a/fisiología , Células Dendríticas/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Tolerancia Inmunológica/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Pyroglyphidae/inmunología , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/inmunología , Receptores CCR7/metabolismoRESUMEN
BACKGROUND: A close association between obesity and asthma has been described. The nature of this association remains elusive, especially with respect to allergic asthma. Controversial findings exist regarding the impact of short-term high-fat diet (HFD) feeding on the development of allergic asthma. OBJECTIVE: To delineate the impact of short-term HFD feeding on the development of experimental allergic asthma. METHODS: Female C57BL/6JRJ mice were fed with a short-term HFD or chow diet (CD) for 12 weeks. Allergic asthma was induced by intraperitoneal OVA/alum sensitization followed by repeated OVA airway challenges. We determined airway hyperresponsiveness (AHR) and pulmonary inflammation by histologic and flow cytometric analysis of immune cells. Furthermore, we assessed the impact of HFD on dendritic cell (DC)-mediated activation of T cells. RESULTS: Female mice showed a mild increase in body weight accompanied by mild metabolic alterations. Upon OVA challenge, CD-fed mice developed strong AHR and airway inflammation, which were markedly reduced in HFD-fed mice. Mucus production was similar in both treatment groups. OVA-induced increases in DC and CD4+ T-cell recruitment to the lungs were significantly attenuated in HFD-fed mice. MHC-II expression and CD40 expression in pulmonary CD11b+ DCs were markedly lower in HFD-fed compared to CD-fed mice, which was associated in vivo with a decreased T helper (Th) 1/17 differentiation and Treg formation without impacting Th2 differentiation. CONCLUSIONS/CLINICAL RELEVANCE: These findings suggest that short-term HFD feeding attenuates the development of AHR, airway inflammation, pulmonary DC recruitment and MHC-II/CD40 expression leading to diminished Th1/17 but unchanged Th2 differentiation. Thus, short-term HFD feeding and associated metabolic alterations may have protective effects in allergic asthma development.
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Alimentación Animal , Asma/inmunología , Asma/prevención & control , Diferenciación Celular/efectos de los fármacos , Grasas de la Dieta/farmacología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Asma/inducido químicamente , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Femenino , RatonesRESUMEN
The biological significance of C5a receptor [(C5aR)2/C5L2], a seven-transmembrane receptor binding C5a and C5adesArg, remains ill-defined. Specific ligation of C5aR2 inhibits C5a-induced ERK1/2 activation, strengthening the view that C5aR2 regulates C5aR1-mediated effector functions. Although C5aR2 and C5aR1 are often coexpressed, a detailed picture of C5aR2 expression in murine cells and tissues is still lacking. To close this gap, we generated a floxed tandem dye (td)Tomato-C5aR2 knock-in mouse that we used to track C5aR2 expression in tissue-residing and circulating immune cells. We found the strongest C5aR2 expression in the brain, bone marrow, and airways. All myeloid-derived cells expressed C5aR2, although with different intensities. C5aR2 expression in blood and tissue neutrophils was strong and homogeneous. Specific ligation of C5aR2 in neutrophils from tdTomato-C5aR2 mice blocked C5a-driven ERK1/2 phosphorylation, demonstrating functionality of C5aR2 in the reporter mice. In contrast to neutrophils, we found tissue-specific differences in C5aR2 expression in eosinophils, macrophages, and dendritic cell subsets. Naive and activated T cells stained negative for C5aR2, whereas B cells from different tissues homogeneously expressed C5aR2. Also, NK cell subsets in blood and spleen strongly expressed C5aR2. Activation of C5aR2 in NK cells suppressed IL-12/IL-18-induced IFN-γ production. Intratracheal IL-33 challenge resulted in decreased C5aR2 expression in pulmonary eosinophils and monocyte-derived dendritic cells. In summary, we provide a detailed map of murine C5aR2 immune cell expression in different tissues under steady-state conditions and upon pulmonary inflammation. The C5aR2 knock-in mouse will help to reliably track and conditionally delete C5aR2 expression in experimental models of inflammation.
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Regulación de la Expresión Génica/inmunología , Leucocitos/inmunología , Neumonía/inmunología , Receptor de Anafilatoxina C5a/inmunología , Animales , Técnicas de Sustitución del Gen , Genes Reporteros/inmunología , Leucocitos/patología , Ratones , Ratones Transgénicos , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Neumonía/genética , Neumonía/patología , Receptor de Anafilatoxina C5a/genéticaRESUMEN
C5a regulates the development of maladaptive immune responses in allergic asthma mainly through the activation of C5a receptor 1 (C5aR1). Yet, the cell types and the mechanisms underlying this regulation are ill-defined. Recently, we described increased C5aR1 expression in lung tissue eosinophils but decreased expression in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs) during the allergic effector phase using a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we determined the role of C5aR1 signaling in neutrophils, moDCs and macrophages for the pulmonary recruitment of such cells and the importance of C5aR1-mediated activation of LysM-expressing cells for the development of allergic asthma. We used LysM-C5aR1 KO mice with a specific deletion of C5aR1 in LysMCre-expressing cells and confirmed the specific deletion of C5aR1 in neutrophils, macrophages and moDCs in the airways and/or the lung tissue. We found that alveolar macrophage numbers were significantly increased in LysM-C5aR1 KO mice. Induction of ovalbumin (OVA)-driven experimental allergic asthma in GFP-C5aR1fl/fl and LysM-C5aR1 KO mice resulted in strong but similar airway resistance, mucus production and Th2/Th17 cytokine production. In contrast, the number of airway but not of pulmonary neutrophils was lower in LysM-C5aR1 KO as compared with GFP-C5aR1fl/fl mice. The recruitment of macrophages, cDCs, moDCs, T cells and type 2 innate lymphoid cells was not altered in LysM-C5aR1 KO mice. Our findings demonstrate that C5aR1 is critical for steady state control of alveolar macrophage numbers and the transition of neutrophils from the lung into the airways in OVA-driven allergic asthma. However, C5aR1 activation of LysM-expressing cells plays a surprisingly minor role in the recruitment and activation of such cells and the development of the allergic phenotype in OVA-driven experimental allergic asthma.
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Asma/patología , Complemento C5a/metabolismo , Pulmón/inmunología , Muramidasa/fisiología , Receptor de Anafilatoxina C5a/fisiología , Linfocitos T/inmunología , Animales , Asma/inducido químicamente , Asma/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Pulmón/metabolismo , Pulmón/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Ovalbúmina/toxicidad , Linfocitos T/metabolismoRESUMEN
C3a exerts multiple biologic functions through activation of its cognate C3a receptor. C3-/- and C3aR-/- mice have been instrumental in defining important roles of the C3a/C3aR axis in the regulation of acute and chronic inflammatory diseases, including ischemia/reperfusion injury, allergic asthma, autoimmune nephritis, and rheumatoid arthritis. Surprisingly little is known about C3aR expression and function in immune and stromal cells. To close this gap, we generated a floxed tandem-dye Tomato (tdTomato)-C3aR reporter knock-in mouse, which we used to monitor C3aR expression in cells residing in the lung, airways, lamina propria (LP) of the small intestine, brain, visceral adipose tissue, bone marrow (BM), spleen, and the circulation. We found a strong expression of tdTomato-C3aR in the brain, lung, LP, and visceral adipose tissue, whereas it was minor in the spleen, blood, BM, and the airways. Most macrophage and eosinophil populations were tdTomato-C3aR+ Interestingly, most tissue eosinophils and some macrophage populations expressed C3aR intracellularly. BM-derived dendritic cells (DCs), lung-resident cluster of differentiation (CD) 11b+ conventional DCs (cDCs) and monocyte-derived DCs, LP CD103+, and CD11b+ cDCs but not pulmonary CD103+ cDCs and splenic DCs were tdTomato-C3aR+ Surprisingly, neither BM, blood, lung neutrophils, nor mast cells expressed C3aR. Similarly, all lymphoid-derived cells were tdTomato-C3aR-, except some LP-derived type 3 innate lymphoid cells. Pulmonary and LP-derived epithelial cells expressed at best minor levels of C3aR. In summary, we provide novel insights into the expression pattern of C3aR in mice. The floxed C3aR knock-in mouse will help to reliably track and conditionally delete C3aR expression in experimental models of inflammation.
Asunto(s)
Genes Reporteros , Receptores Acoplados a Proteínas G/genética , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Complemento C3a/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Expresión Génica , Técnicas de Sustitución del Gen , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Bazo/inmunología , Bazo/metabolismoRESUMEN
C5a drives airway constriction and inflammation during the effector phase of allergic asthma, mainly through the activation of C5a receptor 1 (C5aR1). Yet, C5aR1 expression on myeloid and lymphoid cells during the allergic effector phase is ill-defined. Recently, we generated and characterized a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we used this reporter strain to monitor C5aR1 expression in airway, pulmonary and lymph node cells during the effector phase of OVA-driven allergic asthma. C5aR1 reporter and wildtype mice developed a similar allergic phenotype with comparable airway resistance, mucus production, eosinophilic/neutrophilic airway inflammation and Th2/Th17 cytokine production. During the allergic effector phase, C5aR1 expression increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs). Surprisingly, expression in neutrophils was not affected. Of note, moDCs but not CD11b+ cDCs from mediastinal lymph nodes (mLN) expressed less C5aR1 than DCs residing in the lung after OVA challenge. Finally, neither CD103+ cDCs nor cells of the lymphoid lineage such as Th2 or Th17-differentiated CD4+ T cells, B cells or type 2 innate lymphoid cells (ILC2) expressed C5aR1 under allergic conditions. Our findings demonstrate a complex regulation pattern of C5aR1 in the airways, lung tissue and mLN of mice, suggesting that the C5a/C5aR1 axis controls airway constriction and inflammation through activation of myeloid cells in all three compartments in an experimental model of allergic asthma.
Asunto(s)
Asma/inmunología , Pulmón/inmunología , Receptor de Anafilatoxina C5a/inmunología , Animales , Asma/patología , Antígeno CD11b/análisis , Antígeno CD11b/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Células Dendríticas/inmunología , Células Dendríticas/patología , Eosinófilos/inmunología , Eosinófilos/patología , Inmunidad Celular , Pulmón/patología , Macrófagos/inmunología , Macrófagos/patología , Mediastino/patología , Ratones , Células Mieloides/inmunología , Células Mieloides/patología , Ovalbúmina/inmunología , Receptor de Anafilatoxina C5a/análisisRESUMEN
Many of the biological properties of C5a are mediated through activation of its receptor (C5aR1), the expression of which has been demonstrated convincingly on myeloid cells, such as neutrophils, monocytes, and macrophages. In contrast, conflicting results exist regarding C5aR1 expression in dendritic cells (DCs) and lymphoid lineage cells. In this article, we report the generation of a floxed GFP-C5aR1 reporter knock-in mouse. Using this mouse strain, we confirmed strong C5aR1 expression in neutrophils from bone marrow, blood, lung, and spleen, as well as in peritoneal macrophages. Further, we show C5aR1 expression in lung eosinophils, lung- and lamina propria-resident and alveolar macrophages, bone marrow-derived DCs, and lung-resident CD11b(+) and monocyte-derived DCs, whereas intestinal and pulmonary CD103(+) DCs stained negative. Also, some splenic NKT cells expressed GFP, whereas naive NK cells and B2 cells lacked GFP expression. Finally, we did not observe any C5aR1 expression in naive or activated CD4(+) Th cells in vitro or in vivo. Mating the floxed GFP-C5aR1 mouse strain with LysMCre mice, we were able to specifically delete C5aR1 in neutrophils and macrophages, whereas C5aR1 expression was retained in DCs. In summary, our findings suggest that C5aR1 expression in mice is largely restricted to cells of the myeloid lineage. The novel floxed C5aR1 reporter knock-in mouse will prove useful to track C5aR1 expression in experimental models of acute and chronic inflammation and to conditionally delete C5aR1 in immune cells.
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Células Mieloides/inmunología , Receptor de Anafilatoxina C5a/biosíntesis , Animales , Separación Celular , Citometría de Flujo , Técnicas de Sustitución del Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Anafilatoxina C5a/análisis , Receptor de Anafilatoxina C5a/inmunologíaRESUMEN
The ketone body ß-hydroxybutyrate (BHB) is an endogenous factor protecting against stroke and neurodegenerative diseases, but its mode of action is unclear. Here we show in a stroke model that the hydroxy-carboxylic acid receptor 2 (HCA2, GPR109A) is required for the neuroprotective effect of BHB and a ketogenic diet, as this effect is lost in Hca2(-/-) mice. We further demonstrate that nicotinic acid, a clinically used HCA2 agonist, reduces infarct size via a HCA2-mediated mechanism, and that noninflammatory Ly-6C(Lo) monocytes and/or macrophages infiltrating the ischemic brain also express HCA2. Using cell ablation and chimeric mice, we demonstrate that HCA2 on monocytes and/or macrophages is required for the protective effect of nicotinic acid. The activation of HCA2 induces a neuroprotective phenotype of monocytes and/or macrophages that depends on PGD2 production by COX1 and the haematopoietic PGD2 synthase. Our data suggest that HCA2 activation by dietary or pharmacological means instructs Ly-6C(Lo) monocytes and/or macrophages to deliver a neuroprotective signal to the brain.
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Ácido 3-Hidroxibutírico/metabolismo , Encéfalo/metabolismo , Dieta Cetogénica , Macrófagos/metabolismo , Monocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Encéfalo/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Niacina/farmacología , Receptores Acoplados a Proteínas G/agonistasRESUMEN
Homocystinuria, a disorder originating in defects in the methionine metabolism, is characterized by an elevated plasma concentration of homocysteine. Most patients have a defect in the cystathionine-beta-synthase, the key enzyme in the conversion of homocysteine to cysteine. Many abnormalities in the connective tissue of patients with homocystinuria resemble those seen in Marfan syndrome, caused by mutations in fibrillin-1. These observations led to the hypothesis that the structure and function of fibrillin-1 is compromised in patients with homocystinuria. To test this hypothesis we produced recombinant human fibrillin-1 fragments spanning the central portion of the molecule (8-Cys/transforming growth factor-beta binding domain 3 to calcium binding EGF domain 22) and extensively analyzed the potential of homocysteine to modify structural and functional properties of these proteins. Circular dichroism spectroscopy revealed moderate changes of their secondary structures after incubation with homocysteine. Equilibrium dialysis demonstrated a number of high affinity calcium binding sites in the tandemly repeated calcium binding epidermal growth factor-like domains 11-22. Calcium binding of homocysteine-modified fragments was completely abolished. Incubation of the recombinant proteins with homocysteine rendered the analyzed calcium binding EGF domains as well as the 8-Cys/transforming growth factor-beta binding domain 3 significantly more susceptible to proteolytic degradation. Furthermore, data were obtained demonstrating that homocysteine can covalently modify fibrillin-1 via disulfide bonds. These data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues of patients with homocystinuria play a major role in the pathogenesis of this disorder.
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Homocisteína/química , Homocistinuria/metabolismo , Homocistinuria/patología , Proteínas de Microfilamentos/química , Secuencia de Aminoácidos , Calcio/química , Calcio/metabolismo , Células Cultivadas , Quimotripsina/farmacología , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/metabolismo , Matriz Extracelular/metabolismo , Fibrilina-1 , Fibrilinas , Fibroblastos/metabolismo , Glicosilación , Humanos , Síndrome de Marfan/metabolismo , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Transfección , Tripsina/farmacologíaRESUMEN
Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. The mRNA levels for the mutation constructs were similar to wild-type levels. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. The overall folds of the mutant polypeptides were indistinguishable from the wild-type as judged by the ultrastructural shape, CD analysis, and reactivity with a specific antibody sensitive for intact disulfide bonds. Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. These results expand and further strengthen the concept that proteolytic degradation of mutated fibrillin-1 might be an important potential mechanism in the pathogenesis of Marfan syndrome and other disorders caused by mutations in fibrillin-1.
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Calcio/metabolismo , Cisteína/genética , Factor de Crecimiento Epidérmico/metabolismo , Proteínas de Microfilamentos/metabolismo , Mutación , Secuencia de Aminoácidos , Western Blotting , Línea Celular , Dicroismo Circular , Cisteína/química , Mapeo Epitopo , Fibrilina-1 , Fibrilinas , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación Missense , Péptidos/química , Plásmidos/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Temperatura , Transfección , Rayos UltravioletaRESUMEN
The biological relevance of vigilin a ubiquitous multi (KH)-domain protein is still barely understood. Investigations over the last years, however, provided evidence for a possible involvement of vigilin in the nucleo-cytoplasmic transport of tRNA and in the subsequent association of tRNA with ribosomes. We therefore investigated the potential association of vigilin with 80S ribosomes. Immunostaining, gel filtration, westernblot analysis of polyribosomes and high salt treatment of 80S ribosomes isolated from fresh human placenta were applied to analyze the possible association of vigilin with ribosomes. Overlay assays were performed to examine whether vigilin is capable of binding to ribosomal proteins. Immunostaining of HEp-2 cells, gel filtration of a cytoplasmic extract of HEp-2 cells and westernblot analysis of isolated 80S ribosomes clearly demonstrate that vigilin is bond to the ribosomal complex. Vigilin detaches from the ribosomal complex under the influence of high salt concentrations. We present data that radioactively labeled human vigilin interacts directly with a subset of ribosomal proteins from both subunits. We were able to narrow down the putative binding region to the C-terminal domain by using vigilin mutant constructs. Therefore our results provide strong evidence that vigilin is bond to the ribosomal complex and underline the hypothesis that vigilin might be involved in the link between tRNA-export and the channeled tRNA-cycle on ribosomes.
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Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Polirribosomas/metabolismo , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Portadoras/genética , Línea Celular , Nucléolo Celular/ultraestructura , Femenino , Expresión Génica , Humanos , Placenta/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Ribosomas/química , Eliminación de Secuencia/genéticaRESUMEN
Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.