RESUMEN
Fibrillin-1 is a major component of the extracellular microfibrils, where it interacts with other extracellular matrix proteins to provide elasticity to connective tissues, and regulates the bioavailability of TGFß family members. A peptide consisting of the C-terminal 140 amino acids of fibrillin-1 has recently been identified as a glucogenic hormone, secreted from adipose tissue during fasting and targeting the liver to release glucose. This fragment, called asprosin, also signals in the hypothalamus to stimulate appetite. Asprosin levels are correlated with many of the pathologies indicative of metabolic syndrome, including insulin resistance and obesity. Previous studies and reviews have addressed the therapeutic potential of asprosin as a target in obesity, diabetes and related conditions without considering mechanisms underlying the relationship between generation of asprosin and expression of the much larger fibrillin-1 protein. Profibrillin-1 undergoes obligatory cleavage at the cell surface as part of its assembly into microfibrils, producing the asprosin peptide as well as mature fibrillin-1. Patterns of FBN1 mRNA expression are inconsistent with the necessity for regulated release of asprosin. The asprosin peptide may be protected from degradation in adipose tissue. We present evidence for an alternative possibility, that asprosin mRNA is generated independently from an internal promoter within the 3' end of the FBN1 gene, which would allow for regulation independent of fibrillin-synthesis and is more economical of cellular resources. The discovery of asprosin opened exciting possibilities for treatment of metabolic syndrome related conditions, but there is much to be understood before such therapies could be introduced into the clinic.
Asunto(s)
Síndrome Metabólico , Humanos , Fibrilina-1/genética , Fibrilina-2 , Fibrilinas , Glucosa , Síndrome Metabólico/genética , Proteínas de Microfilamentos/genética , Obesidad/genética , ARN Mensajero , Adipoquinas/genéticaRESUMEN
Fibrillin-1 is a large glycoprotein encoded by the FBN1 gene in humans. It provides strength and elasticity to connective tissues and is involved in regulating the bioavailability of the growth factor TGFß. Mutations in FBN1 may be associated with depleted or abnormal adipose tissue, seen in some patients with Marfan syndrome and lipodystrophies. As this lack of adipose tissue does not result in high morbidity or mortality, it is generally under-appreciated, but is a cause of psychosocial problems particularly to young patients. We examined the role of fibrillin-1 in adipogenesis. In inbred mouse strains we found significant variation in the level of expression in the Fbn1 gene that correlated with variation in several measures of body fat, suggesting that mouse fibrillin-1 is associated with the level of fat tissue. Furthermore, we found that FBN1 mRNA was up-regulated in the adipose tissue of obese women compared to non-obese, and associated with an increase in adipocyte size. We used human mesenchymal stem cells differentiated in culture to adipocytes to show that fibrillin-1 declines after the initiation of differentiation. Gene expression results from a similar experiment (available through the FANTOM5 project) revealed that the decline in fibrillin-1 protein was paralleled by a decline in FBN1 mRNA. Examination of the FBN1 gene showed that the region commonly affected in FBN1-associated lipodystrophy is highly conserved both across the three human fibrillin genes and across genes encoding fibrillin-1 in vertebrates. These results suggest that fibrillin-1 is involved as the undifferentiated mesenchymal stem cells transition to adipogenesis but then declines as the developing adipocytes take on their final phenotype. Since the C-terminal peptide of fibrillin-1 is a glucogenic hormone, individuals with low fibrillin-1 (for example with FBN1 mutations associated with lipodystrophy) may fail to differentiate adipocytes and/or to accumulate adipocyte lipids, although this still needs to be shown experimentally.
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Adipogénesis/genética , Diferenciación Celular/genética , Fibrilina-1/genética , Células Madre Mesenquimatosas/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Humanos , Lipodistrofia/genética , Lipodistrofia/fisiopatología , Masculino , Síndrome de Marfan/genética , Síndrome de Marfan/fisiopatología , Ratones , Mutación , Fenotipo , ARN Mensajero/genética , Caracteres SexualesRESUMEN
The HMWABC system of non-typeable Haemophilus influenzae (NTHi) encodes the HMWA adhesin glycoprotein, which is glycosylated by the HMWC glycosyltransferase. HMWC is a cytoplasmic N-glycosyltransferase, homologues of which are widespread in the Pasteurellaceae. We developed an assay for nonbiased detection of glycoproteins in NTHi based on metabolic engineering of the Leloir pathway and growth in media containing radiolabelled monosaccharides. The only glycoprotein identified in NTHi by this assay was HMWA. However, glycoproteomic analyses ex vivo in Escherichia coli showed that HMWC of NTHi was a general glycosyltransferase capable of glycosylating selected asparagines in proteins other than its HMWA substrate, including Asn78 in E. coli 30S ribosomal protein S5. The equivalent residue in S5 homologues in H. influenzae or other sequenced Pasteurellaceae genomes is not asparagine, and these organisms also showed significantly fewer than expected potential sites of glycosylation in general. Expression of active HMWC in E. coli resulted in growth inhibition compared with expression of inactive enzyme, consistent with glycosylation by HMWC detrimentally affecting the function of some E. coli proteins. Together, this supports the presence of a selective pressure in the Pasteurellaceae against glycosylation sites that would be modified by the general N-glycosyltransferase activity of HMWC.
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Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Glucosiltransferasas/metabolismo , Glicoproteínas/metabolismo , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/enzimología , Adhesinas Bacterianas/análisis , Secuencia de Aminoácidos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Clonación Molecular , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Glucosiltransferasas/genética , Glicoproteínas/análisis , Glicosilación , Haemophilus influenzae/química , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Humanos , Redes y Vías Metabólicas , Modelos Moleculares , Datos de Secuencia Molecular , ProteómicaRESUMEN
The fibrillins and latent transforming growth factor binding proteins (LTBPs) form a superfamily of extracellular matrix (ECM) proteins characterized by the presence of a unique domain, the 8-cysteine transforming growth factor beta (TGFß) binding domain. These proteins are involved in the structure of the extracellular matrix and controlling the bioavailability of TGFß family members. Genes encoding these proteins show differential expression in mesenchymal cell types which synthesize the extracellular matrix. We have investigated the promoter regions of the seven gene family members using the FANTOM5 CAGE database for human. While the protein and nucleotide sequences show considerable sequence similarity, the promoter regions were quite diverse. Most genes had a single predominant transcription start site region but LTBP1 and LTBP4 had two regions initiating different transcripts. Most of the family members were expressed in a range of mesenchymal and other cell types, often associated with use of alternative promoters or transcription start sites within a promoter in different cell types. FBN3 was the lowest expressed gene, and was found only in embryonic and fetal tissues. The different promoters for one gene were more similar to each other in expression than to promoters of the other family members. Notably expression of all 22 LTBP2 promoters was tightly correlated and quite distinct from all other family members. We located candidate enhancer regions likely to be involved in expression of the genes. Each gene was associated with a unique subset of transcription factors across multiple promoters although several motifs including MAZ, SP1, GTF2I and KLF4 showed overrepresentation across the gene family. FBN1 and FBN2, which had similar expression patterns, were regulated by different transcription factors. This study highlights the role of alternative transcription start sites in regulating the tissue specificity of closely related genes and suggests that this important class of extracellular matrix proteins is subject to subtle regulatory variations that explain the differential roles of members of this gene family.
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Perfilación de la Expresión Génica , Proteínas de Unión a TGF-beta Latente/genética , Células Madre Mesenquimatosas/metabolismo , Proteínas de Microfilamentos/genética , Línea Celular , Elementos de Facilitación Genéticos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Humanos , Factor 4 Similar a Kruppel , Proteínas de Unión a TGF-beta Latente/metabolismo , Proteínas de Microfilamentos/metabolismo , Familia de Multigenes , Especificidad de Órganos , Regiones Promotoras GenéticasRESUMEN
Fibrillins and latent transforming growth factor ß binding proteins (LTBPs) are components of the extracellular matrix of connective tissue. While fibrillins are integral to the 10nm microfibrils, and often associated with elastin, all family members are likely to have an additional role in regulating the bioavailability of transforming growth factor ß (TGBß). Both fibrillins and LTBPs are large glycoproteins, containing a series of calcium binding epidermal growth factor domains as well as a number of copies of a unique 8 cysteine domain found only in this protein superfamily. There are three mammalian fibrillins and four LTBPs. Fibrillin monomers link head to tail in microfibrils which can then form two and three dimensional structures. In some tissues elastin is recruited to the fibrillin microfibrils to provide elasticity to the tissue. LTBPs are part of the TGBß large latent complex which sequesters TGBß in the extracellular matrix. Fibrillin-1 appears to bind to LTBPs to assist in this process and is thus involved in regulating the bioavailability of TGBß. Mutation of fibrillin genes results in connective tissue phenotypes which reflect both the increased level of active TGBß and the structural failure of the extracellular matrix due to the absence or abnormality of fibrillin protein. Fibrillinopathies include Marfan syndrome, familial ectopia lentis, familial thoracic aneurysm (mutations of FBN1) and congenital contractural arachnodactyly (mutation of FBN2). There are no diseases currently associated with mutation of FBN3 in humans, and this gene is no longer active in rodents. Expression patterns of fibrillin genes are consistent with their role in extracellular matrix structure of connective tissue. FBN1 expression is high in most cell types of mesenchymal origin, particularly bone. Human and mouse FBN2 expression is high in fetal cells and has more restricted expression in mesenchymal cell types postnatally. FBN3 is expressed early in development (embryonic and fetal tissues) in humans. The fibrillins are thus important in maintaining the structure and integrity of the extracellular matrix and, in combination with their sequence family members the LTBPs, also contribute to the regulation of the TGFß family of major growth factors.
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Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Familia de Multigenes , Animales , Enfermedades del Tejido Conjuntivo/genética , Enfermedades del Tejido Conjuntivo/metabolismo , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Proteínas de Unión a TGF-beta Latente/metabolismo , MutaciónRESUMEN
This article contains data related to the research article entitled "Expression of FBN1 during adipogenesis: relevance to the lipodystrophy phenotype in Marfan syndrome and related conditions" [1]. The article concerns the expression of FBN1, the gene encoding the extracellular matrix protein fibrillin-1, during adipogenesis in vitro and in relation to adipose tissue in vivo. The encoded protein has recently been shown to produce a short glucogenic peptide hormone, (Romere et al., 2016) [2], and this gene is therefore a key gene for regulating blood glucose levels. FBN1 and coexpressed genes were examined in mouse strains and in human cells undergoing adipogenesis. The data show the genes that were coexpressed with FBN1, including genes coding for other connective tissue proteins and the proteases that modify them and for the transcription factors that control their expression. Data analysed were derived from datasets available in the public domain and the analysis highlights the utility of such datasets for ongoing analysis and hence reduction in the use of experimental animals.
RESUMEN
UNLABELLED: The aim of this study was to do an initial assessment of the usefulness of 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-(18)F-fluoroethyl)nortropane ((18)F-FECNT) PET scanning in determining in vivo brain dopamine transporter (DAT) density in healthy humans and subjects with Parkinson's disease (PD). METHODS: We investigated 6 neurologically healthy subjects and 5 PD patients: 2 with mild unilateral disease, 1 with mild-to-moderate bilateral disease, and 2 with moderately severe bilateral disease. The healthy subjects underwent a 3-h PET scan (26 frames) and the PD subjects underwent a 2-h PET scan (23 frames) while (18)F-FECNT was being injected over the first 5 min of the scan. Arterial blood samples were taken throughout scanning for well-counter and metabolite analysis to determine the presence of possible active metabolites. The scans were reconstructed; then we placed spheric regions of interest in the caudate nuclei, putamena, thalami, brain stem, cerebellum, and occipital cortex of each subject. The radioactivity level in each region was calculated for each frame of a subject's PET scan. Then we calculated target tissue-to-cerebellum ratios for each time frame. RESULTS: The analysis of arterial blood samples revealed that metabolism of the tracer was rapid. The ether-extractable component of the arterial input was >98% pure (18)F-FECNT. The caudate nucleus and putamen exhibited the highest uptake and prolonged retention of the radioligand. They both attained maximum uptake at approximately 90 min, with the healthy subjects' average caudate- and putamen-to-cerebellum ratios (+/-SD) at that time being 9.0 +/- 1.2 and 7.8 +/- 0.7, respectively. The maximal caudate-to-cerebellum ratios for the healthy subjects ranged from 7.6 to 10.5 and their maximal putamen-to-cerebellum ratios ranged from 7.1 to 9.3. The 2 early-stage, unilateral PD patients had, at 90 min, an average right caudate-to-cerebellum ratio of 5.3 +/- 1.1 and a left ratio of 5.9 +/- 0.7 and an average right putamen-to cerebellum ratio of 2.8 +/- 0.1 and a left ratio of 3.0 +/- 0.6. The late-stage PD patients had, at 90 min, an average right caudate-to-cerebellum ratio of 3.7 +/- 0.4 and a left ratio of 3.9 +/- 0 and an average right putamen-to cerebellum ratio of 1.8 +/- 0.1 and a left ratio of 1.8 +/- 0. CONCLUSION: These results indicate that (18)F-FECNT is an excellent candidate radioligand for in vivo imaging of the DAT system in humans. It has a much higher affinity for DAT than for the serotonin transporter and yields the highest peak striatum-to-cerebellum ratios and has among the most favorable kinetics of (18)F-radiolabeled DAT ligands. Having picked up presymptomatic changes in the hemisphere opposite the unaffected side of the body in our early-stage (unilateral) PD patients, it appears that, like other DAT radioligands, it may be able to identify presymptomatic PD.
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Glicoproteínas de Membrana , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso , Nortropanos/farmacocinética , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/metabolismo , Tomografía Computarizada de Emisión/métodos , Adulto , Anciano , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Estudios de Factibilidad , Femenino , Radioisótopos de Flúor/farmacocinética , Humanos , Ligandos , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Radiofármacos/farmacocinéticaRESUMEN
INTRODUCTION: Fluorine-18 labeled 2ß-carbomethoxy-3ß-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane ([(18)F]FECNT) binds reversibly to the dopamine transporter (DAT) with high selectivity. [(18)F]FECNT has been used extensively in the quantification of DAT occupancy in non-human primate brain and can distinguish between Parkinson's and healthy controls in humans. The purpose of this work was to develop a compartment model to characterize the kinetics of [(18)F]FECNT for quantification of DAT density in healthy human brain. METHODS: Twelve healthy volunteers underwent 180 min dynamic [(18)F]FECNT PET imaging including sampling of arterial blood. Regional time-activity curves were extracted from the caudate, putamen and midbrain including a reference region placed in the cerebellum. Binding potential, BPND, was calculated for all regions using kinetic parameters estimated from compartmental and Logan graphical model fits to the time-activity data. Simulations were performed to determine whether the compartment model could reliably fit time-activity data over a range of BPND values. RESULTS: The kinetics of [(18)F]FECNT were well-described by the reversible 2-tissue arterial input and full reference tissue compartment models. Calculated binding potentials in the caudate, putamen and midbrain were in good agreement between the arterial input model, reference tissue model and the Logan graphical model. The distribution volume in the cerebellum did not reach a plateau over the duration of the study, which may be a result of non-specific binding in the cerebellum. Simulations that included non-specific binding show that the reference and arterial input models are able to estimate BPND for DAT densities well below that observed in normal volunteers. CONCLUSION: The kinetics of [(18)F]FECNT in human brain are well-described by arterial input and reference tissue compartment models. Measured and simulated data show that BPND calculated with reference tissue model is proportional to BPND calculated from the arterial input model.
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Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Voluntarios Sanos , Nortropanos , Tomografía de Emisión de Positrones/métodos , Adulto , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Modelos Biológicos , Nortropanos/metabolismoRESUMEN
The antitumor agent sulofenur (LY186641), which has shown promising activity against a wide range of cancers, causes hemolytic anemia and methemoglobinemia at dose-limiting toxicities. The antitumor and toxicological mechanism(s) of action of the drug is (are) not well understood, but unlike other antineoplastic agents, sulofenur does not interfere with DNA, RNA, or protein synthesis, or with polynucleotide function. In the present study, we evaluated the hypothesis that sulofenur undergoes bioactivation in vivo to generate p-chlorophenyl isocyanate (CPIC), which could carbamoylate biological macromolecules directly or form a conjugate with glutathione (GSH) which would serve as a latent form of CPIC. The objectives of this study, therefore, were to determine if the GSH and N-acetylcysteine conjugates of CPIC were excreted into bile and urine, respectively, after an i.p. dose of sulofenur to rats. In addition, the chemical stability and thiol exchange properties of these S-linked conjugates were determined. The results of this study indicate that sulofenur does undergo metabolism in vivo to yield the GSH conjugate of CPIC, and that this conjugation reaction is reversible and subject to thiol exchange in buffered aqueous solution (pH 7.4, 37 degrees C). In contrast, sulofenur itself was stable under these same conditions, even in the presence of GSH and glutathione-S-transferase (GST), thus raising the possibility that bioactivation of sulofenur is necessary for liberation of CPIC. These findings suggest that the generation of this isocyanate in vivo and subsequent carbamoylation of biological macromolecules may play a role in the toxicity and/or antitumor activity of sulofenur and related diarylsulfonylureas.
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Antineoplásicos/metabolismo , Glutatión/metabolismo , Isocianatos/metabolismo , Compuestos de Sulfonilurea/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The disposition and metabolism of rofecoxib, a selective cyclooxygenase-2 inhibitor, were examined in healthy human subjects and in cholecystectomy patients. After oral administration of [(14)C]rofecoxib (125 mg, 100 micro Ci) to healthy subjects, the mean concentrations of total radioactivity and rofecoxib in plasma as a function of time indicated that the t(max) was achieved at 9 h postdose. After t(max), levels of both radioactivity and rofecoxib decreased in a parallel, exponential fashion (effective t(1/2) approximately equal 17 h). A similar result was obtained after oral administration of [(14)C]rofecoxib (142 mg, 100 micro Ci) to cholecystectomy patients equipped with an L-tube. In healthy subjects, radioactivity was recovered predominantly from the urine (71.5% of dose), with a small amount excreted in feces (14.2%). In patients with an L-tube, half the radioactive dose was recovered in feces, with a lesser amount excreted in urine (28.8%) and a negligible fraction in bile (1.8%). Rofecoxib underwent extensive metabolism in humans, and very little parent drug was recovered unchanged in urine (<1%). Products resulting from both oxidative and reductive pathways were identified by a combination of (1)H NMR and liquid chromatography-tandem mass spectrometry analyses, and included rofecoxib-3',4'-trans-dihydrodiol, 4'-hydroxyrofecoxib-O-beta-D-glucuronide, diastereomeric 5-hydroxyrofecoxib-O-beta-D-glucuronide conjugates, 5-hydroxyrofecoxib, rofecoxib-erythro-3,4-dihydrohydroxy acid, and rofecoxib-threo-3,4-dihydrohydroxy acid. Interconversion of rofecoxib and 5-hydroxyrofecoxib appeared not to be a quantitatively important pathway of rofecoxib disposition in human subjects, in contrast to previous findings in rats.