RESUMEN
The fibrinolytic system is composed of the protease plasmin, its precursor plasminogen and their respective activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), counteracted by their inhibitors, plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), protein C inhibitor (PCI), thrombin activable fibrinolysis inhibitor (TAFI), protease nexin 1 (PN-1) and neuroserpin. The action of plasmin is counteracted by α2-antiplasmin, α2-macroglobulin, TAFI, and other serine protease inhibitors (antithrombin and α2-antitrypsin) and PN-1 (protease nexin 1). These components are essential regulators of many physiologic processes. They are also involved in the pathogenesis of many disorders. Recent advancements in our understanding of these processes enable the opportunity of drug development in treating many of these disorders.
Asunto(s)
Fibrinolisina , Fibrinólisis , Fibrinolisina/metabolismo , Fibrinólisis/fisiología , Plasminógeno/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Nexinas de Proteasas , Activador de Tejido Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , alfa 2-AntiplasminaRESUMEN
Extracellular signal-regulated kinase (ERK), also known as classical mitogen-activated protein kinase, plays critical roles in cell regulation. ERK is activated through phosphorylation by a cascade of protein kinases including MEK. Various ligands activate the MEK/ERK pathway through receptor-dependent cell signaling. In cultured cells, many ligands such as growth factors, hormones, cytokines and vasoactive peptides elicit transient activation of MEK/ERK, often peaking at ~10â¯min after the cell treatment. Here, we describe a novel biological event, in which ligand-mediated cell signaling results in the dephosphorylation of MEK/ERK. Neuromedin N and neurotensin, peptides derived from the same precursor polypeptide, elicit cell signaling through the neurotensin receptors. In cultured human pulmonary artery smooth muscle cells (PASMCs), but not in human pulmonary artery endothelial cells (PAECs), we found that both neuromedin N and neurotensin promoted the dephosphorylation of ERK and MEK. Human PASMCs were found to express neurotensin receptor (NTR)-1, -2 and -3, while human PAECs only express NTR3. Neuromedin N-mediated dephosphorylation was suppressed by small chemical inhibitors of protein phosphatase 1/2A and peptidyl-prolyl isomerase. Transmission electron microscopy showed the formation of endocytic vesicles in response to neuromedin N treatment, and dephosphorylation did not occur when sorting nexin 9, a critical regulator of the endocytic vesicle formation, was knocked down. We conclude that neuromedin N and neurotensin elicit a unique dephosphorylation signaling in the MEK/ERK pathway that is regulated by endocytosis. Considering the pathophysiological importance of the MEK/ERK pathway, this discovery of the dephosphorylation mechanism should advance the field of cell signaling.
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Células Endoteliales/metabolismo , Miocitos del Músculo Liso/metabolismo , Neurotensina/fisiología , Fragmentos de Péptidos/fisiología , Arteria Pulmonar/metabolismo , Células Endoteliales/citología , Humanos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Sistema de Señalización de MAP Quinasas , Miocitos del Músculo Liso/citología , Proteínas Nucleares , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Nexinas de Proteasas/metabolismo , Arteria Pulmonar/citología , Proteínas de Unión al ARN , Receptores de Neurotensina/metabolismoRESUMEN
Cultures of glial cells and fibroblasts allowed and lead to the identification SERPINE2/Protease Nexin-1 (SERPINE2/PN-1). Cellular, biochemical, immunological and molecular characterization substantiated its variable expression in many organs as a function of development, adult stages, pathological situations or following injury. It is not a circulating serpin, but as other members of the family, its target specificity is influenced by components of the extracellular matrix. The challenges are to identify where and when SERPINE2/PN-1 modulatory action becomes crucial or even possibly specific in a mosaic of feasible in vivo impacts. Data providing correlations are not sufficient to satisfy this aim. Genetically modified mice, or tissue derived thereof, provide interesting in vivo models to identify and study the relevance of this serpin. This review will highlight sometimes-intriguing results indicating a crucial impact of SERPINE2/PN-1, especially in the vasculature, the nervous system or the behavior of cancer cells in vivo. Data presently available will be discussed in an attempt to define general trends in the diversity of SERPINE2/PN-1 modes of action in vivo.
Asunto(s)
Nexinas de Proteasas/metabolismo , Serpina E2/metabolismo , Animales , Inhibidores Enzimáticos/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Ligandos , Receptores de Superficie Celular/metabolismoRESUMEN
Phosphoinositides are a type of cellular phospholipid that regulate signaling in a wide range of cellular and physiological processes through the interaction between their phosphorylated inositol head group and specific domains in various cytosolic proteins. These lipids also influence the activity of transmembrane proteins. Aberrant phosphoinositide signaling is associated with numerous diseases, including cancer, obesity, and diabetes. Thus, identifying phosphoinositide-binding partners and the aspects that define their specificity can direct drug development. However, current methods are costly, time-consuming, or technically challenging and inaccessible to many laboratories. We developed a method called PLIF (for "protein-lipid interaction by fluorescence") that uses fluorescently labeled liposomes and tethered, tagged proteins or peptides to enable fast and reliable determination of protein domain specificity for given phosphoinositides in a membrane environment. We validated PLIF against previously known phosphoinositide-binding partners for various proteins and obtained relative affinity profiles. Moreover, PLIF analysis of the sorting nexin (SNX) family revealed not only that SNXs bound most strongly to phosphatidylinositol 3-phosphate (PtdIns3P or PI3P), which is known from analysis with other methods, but also that they interacted with other phosphoinositides, which had not previously been detected using other techniques. Different phosphoinositide partners, even those with relatively weak binding affinity, could account for the diverse functions of SNXs in vesicular trafficking and protein sorting. Because PLIF is sensitive, semiquantitative, and performed in a high-throughput manner, it may be used to screen for highly specific protein-lipid interaction inhibitors.
Asunto(s)
Fosfatos de Fosfatidilinositol/química , Nexinas de Proteasas/química , Transducción de Señal , Animales , Ratones , Fosfatos de Fosfatidilinositol/metabolismo , Nexinas de Proteasas/metabolismoRESUMEN
Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families.
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Proteínas del Citoesqueleto/genética , Dineínas/antagonistas & inhibidores , Síndrome de Kartagener/etiología , Complejos Multiproteicos/antagonistas & inhibidores , Mutación/genética , Proteínas de Neoplasias/genética , Nexinas de Proteasas/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Adulto , Animales , Western Blotting , Niño , Cilios/fisiología , Dineínas/genética , Exoma/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Síndrome de Kartagener/patología , Masculino , Proteínas de la Membrana , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Complejos Multiproteicos/genética , Mucosa Nasal/citología , Mucosa Nasal/metabolismo , Óxido Nítrico/análisis , Linaje , Fenotipo , Pronóstico , Nexinas de Proteasas/genética , Sistema Respiratorio , Adulto JovenAsunto(s)
Encéfalo/fisiología , Hemostasis/fisiología , Accidente Cerebrovascular/prevención & control , Accidente Cerebrovascular/terapia , Trombosis/complicaciones , Animales , Antitrombina III/metabolismo , Coagulación Sanguínea , Encéfalo/fisiopatología , Hemorragia Cerebral/patología , Modelos Animales de Enfermedad , Epoprostenol/metabolismo , Humanos , Microcirculación , Óxido Nítrico/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Nexinas de Proteasas/metabolismo , Trombomodulina/metabolismo , Tromboplastina/metabolismo , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Axonemal dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo-electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 dynein and structurally appeared to connect with the nexin-dynein regulatory complex. In axonemes from mutants that lack both the outer dynein arms and the MIA complex, I1 dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 dynein. The MIA complex appears to regulate I1 dynein and possibly outer arm dyneins, which are both essential for normal motility.
Asunto(s)
Movimiento Celular , Chlamydomonas reinhardtii/citología , Cilios/metabolismo , Secuencia Conservada , Dineínas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Axonema/metabolismo , Secuencia de Bases , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestructura , Cilios/ultraestructura , Dineínas/química , Genes de Plantas , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Nexinas de Proteasas/metabolismo , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Secuencias Repetitivas de AminoácidoRESUMEN
PURPOSE: The purpose of this study was to identify genes enriched in breast cancer stroma, assess the stromal gene expression differences between estrogen receptor (ER) -positive and -negative cancers, and separately determine their prognostic value in these two subtypes of breast cancers. METHODS: We compared gene expression profiles of pairs of fine-needle (stroma-poor) and core-needle (stroma-rich) biopsies from 37 cancers to identify stroma-associated genes. We defined stromal metagenes and tested their prognostic values in 684 node-negative patients who received no systemic adjuvant therapy and 259 tamoxifen-treated patients. RESULTS: We identified 293 probe sets overexpressed in core biopsies; these included five highly coexpressed gene clusters (metagenes) corresponding to immune functions and extracellular matrix components. These genes showed quantitative and qualitative differences between ER-positive and ER-negative cancers. A B-cell/plasma cell metagene showed strong prognostic value in ER-positive highly proliferative cancers, a lesser prognostic value in ER-negative cancers, and no prognostic value in ER-positive cancers with low proliferation. The hazard ratio for distant relapse in the lowest compared with the highest tertile of the pooled prognostic data set was 4.29 (95% CI, 2.04 to 9.01; P = .001) in ER-positive cancers and 3.34 (95% CI, 1.60 to 6.97; P = .001) in ER-negative cancers. This remained significant in multivariate analysis including routine variables and other genomic prognostic scores. As a result of quantitative differences in this metagene between ER-positive and ER-negative cancers, different thresholds apply in the two subgroups. Other stromal metagenes had inconsistent prognostic value. CONCLUSION: Among ER-negative and ER-positive highly proliferative cancers, a subset of tumors with high expression of a B-cell/plasma cell metagene carries a favorable prognosis.
Asunto(s)
Neoplasias de la Mama/genética , Receptores de Estrógenos/genética , Precursor de Proteína beta-Amiloide/genética , Antineoplásicos Hormonales/uso terapéutico , Linfocitos B/patología , Biopsia con Aguja Fina , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Distribución de Chi-Cuadrado , Colágeno Tipo IV/genética , Proteínas de la Matriz Extracelular , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Metagenoma/genética , Recurrencia Local de Neoplasia , Proteínas de Transferencia de Fosfolípidos/genética , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Nexinas de Proteasas , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Superficie Celular/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Análisis de Supervivencia , Tamoxifeno/uso terapéuticoRESUMEN
Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, ß-amyloid (Aß) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aß sequence was replaced by the human Aß. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aß pathogenesis.
Asunto(s)
Alelos , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Unión Neuromuscular/metabolismo , Receptores de Superficie Celular/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Mutación del Sistema de Lectura , Técnicas de Sustitución del Gen , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Unión Neuromuscular/genética , Unión Neuromuscular/patología , Nexinas de Proteasas , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genéticaRESUMEN
Islet amyloid polypeptide (IAPP, amylin) is the major protein component of the islet amyloid deposits associated with type 2 diabetes. The polypeptide lacks a well-defined structure in its monomeric state but readily assembles to form amyloid. Amyloid fibrils formed from IAPP, intermediates generated in the assembly of IAPP amyloid, or both are toxic to ß-cells, suggesting that islet amyloid formation may contribute to the pathology of type 2 diabetes. There are relatively few reported inhibitors of amyloid formation by IAPP. Here we show that the tea-derived flavanol, (-)-epigallocatechin 3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] (EGCG), is an effective inhibitor of in vitro IAPP amyloid formation and disaggregates preformed amyloid fibrils derived from IAPP. The compound is thus one of a very small set of molecules which have been shown to disaggregate IAPP amyloid fibrils. Fluorescence-detected thioflavin-T binding assays and transmission electron microscopy confirm that the compound inhibits unseeded amyloid fibril formation as well as disaggregates IAPP amyloid. Seeding studies show that the complex formed by IAPP and EGCG does not seed amyloid formation by IAPP. In this regard, the behavior of IAPP is similar to the reported interactions of Aß and α-synuclein with EGCG. Alamar blue assays and light microscopy indicate that the compound protects cultured rat INS-1 cells against IAPP-induced toxicity. Thus, EGCG offers an interesting lead structure for further development of inhibitors of IAPP amyloid formation and compounds that disaggregate IAPP amyloid.
Asunto(s)
Amiloide/antagonistas & inhibidores , Amiloide/metabolismo , Amiloide/química , Precursor de Proteína beta-Amiloide , Animales , Benzotiazoles , Catequina/análogos & derivados , Técnicas de Cultivo de Célula , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Flavonoides , Polipéptido Amiloide de los Islotes Pancreáticos , Microscopía Electrónica de Transmisión , Fenoles , Polifenoles , Nexinas de Proteasas , Ratas , Receptores de Superficie Celular , Tiazoles , alfa-Sinucleína/análisis , alfa-Sinucleína/metabolismoRESUMEN
We discovered a nonpeptidic compound, TAK-070, that inhibited BACE1, a rate-limiting protease for the generation of Abeta peptides that are considered causative for Alzheimer's disease (AD), in a noncompetitive manner. TAK-070 bound to full-length BACE1, but not to truncated BACE1 lacking the transmembrane domain. Short-term oral administration of TAK-070 decreased the brain levels of soluble Abeta, increased that of neurotrophic sAPPalpha by approximately 20%, and normalized the behavioral impairments in cognitive tests in Tg2576 mice, an APP transgenic mouse model of AD. Six-month chronic treatment decreased cerebral Abeta deposition by approximately 60%, preserving the pharmacological efficacy on soluble Abeta and sAPPalpha levels. These results support the feasibility of BACE1 inhibition with a noncompetitive inhibitor as disease-modifying as well as symptomatic therapy for AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Compuestos de Bifenilo/farmacología , Encéfalo/efectos de los fármacos , Trastornos del Conocimiento/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Naftalenos/farmacología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Compuestos de Bifenilo/química , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Estudios de Factibilidad , Femenino , Humanos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Transgénicos , Naftalenos/química , Nexinas de Proteasas , Distribución Aleatoria , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Resultado del TratamientoRESUMEN
Matrix metalloproteinase-9 (MMP-9) expression is known to enhance the invasion and metastasis of tumor cells. In previous work based on a proteomic screen, we identified the serpin protease nexin-1 (PN-1) as a potential target of MMP-9. Here, we show that PN-1 is a substrate for MMP-9 and establish a link between PN-1 degradation by MMP-9 and regulation of invasion. PN-1 levels increased in prostate carcinoma cells after downregulation of MMP-9 and in tissues of MMP-9-deficient mice, consistent with PN-1 degradation by MMP-9. We identified three MMP-9 cleavage sites in PN-1 and showed that mutations in those sites made PN-1 more resistant to MMP-9. Urokinase plasminogen activator (uPA) is inhibited by PN-1. MMP-9 augmented uPA activity in the medium of PC3-ML cells by degrading PN-1. Prostate cancer cells, overexpressing PN-1 or treated with MMP-9 shRNA, had reduced cell invasion in Matrigel. PN-1 siRNA restored uPA activity and the invasive capacity. PN-1 mutated in the serpin inhibitory domain, the reactive center loop, failed to inhibit uPA and to reduce Matrigel invasion. This study shows a novel molecular pathway in which MMP-9 regulates uPA activity and tumor cell invasion through cleavage of PN-1.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Receptores de Superficie Celular/metabolismo , Serpinas/metabolismo , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica , Nexinas de Proteasas , ARN Interferente Pequeño/genética , Serpina E2 , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
BACE-1 is one of the aspartic proteases involved in the cleavage of beta amyloid peptide, an initial step in the formation of amyloid plaques whose toxicity induces neuron death in Alzheimer's disease patients. One of the central issues in the search of novel BACE-1 inhibitors is the optimum pH for the binding of inhibitors to the enzyme. It is known that the enzyme has optimal catalytic activity at acidic pH, while cell active inhibitors may bind optimally at higher pH. In this work we determine the effect of the pH on the affinities of a set of inhibitors, with a variety of chemical motifs, for the ectodomain region of BACE-1 by a surface plasmon resonance (SPR) biosensor based assay. In order to understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we have calculated the titration curves for a set of BACE-1 ligand complexes. The results indicate that the pK(a) values of the titratable residues of the protein depend on the nature of the ligand involved, in disagreement with previous work. The enzyme-inhibitor structures with the resulting protonation states at pH values 4.5 and 7.4 served as the starting point for the prediction of the pH-dependent binding ranking. Our calculations reproduced the entire affinity ranking observed upon pH increase and most of the binding trends among inhibitors, especially at low pH. Finally, our cell-based assays indicate a possible correlation between high inhibitor affinity at both acidic and neutral pH values, with optimal cell response, a result that may open new venues for the search of potent BACE-1 inhibitors that are active at the cellular level.
Asunto(s)
Inhibidores Enzimáticos/química , Fenómenos Físicos , Precursor de Proteína beta-Amiloide , Inhibidores Enzimáticos/farmacología , Humanos , Ligandos , Nexinas de Proteasas , Estructura Terciaria de Proteína , Receptores de Superficie CelularRESUMEN
OBJECTIVE: To investigate the genetic polymorphisms of rs2229338 and rs12218 loci of serum amyloid protein A1 (SAA1) gene in healthy Chinese Han and Uighur populations of Xinjiang. METHODS: The genotypes of the SAA1 gene were detected in 316 Uighur and 362 Han healthy individuals by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). RESULTS: The genotype distributions of both populations were in the Hardy-Weinberg equilibrium (both P>0.05). The frequencies of AA, AG and GG genotypes of the rs2229338 locus were 76.6%, 23.4%, and 0 in the Uighurs, and 91.7%, 7.7% and 0.6% in the Hans. There was significant difference in distribution of genotypes between the two populations (P<0.01). The frequencies of CC, CT and TT genotypes of the rs12218 locus were 10.1%, 47.5%, and 42.4% in Uighurs, and 3.3%, 34.3% and 62.4% in Hans. There was also significant difference in distribution of genotypes between the two populations (P<0.01). The A-C and G-T haplotypes were more frequent in the Uighur but the A-T haplotype was more common in the Han population, respectively (both P<0.01). CONCLUSION: The mutational frequencies of the tagging SNPs in rs2229338 and rs12218 loci of theSAA1 gene in the Uighurs may be higher than those in Hans.
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Alelos , Pueblo Asiatico/genética , Frecuencia de los Genes/ética , Haplotipos/ética , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Proteína Amiloide A Sérica/genética , Amiloide/genética , Amiloide/metabolismo , Etnicidad/genética , Genotipo , Haplotipos/genética , Humanos , Polimorfismo de Longitud del Fragmento de Restricción/genética , Nexinas de Proteasas/genéticaRESUMEN
Cerebral amyloid angiopathy is a common feature in Alzheimer's disease (AD), which is characterized by amyloid deposit around brain vessels including capillaries. The origin of the amyloid protein of CAA remains controversial. In our work, we provide data to show that primary umbilical vein endothelial cells (HUVEC) harbor APP processing secretases and can produce Abeta(42) under starvation. Starvation can increase the secretion of Abeta(42) by altering the expression of beta-secretases (BACE1) and gamma-secretases (APH and PEN2). This process is regulated by macroautophagy. Suppression of macroautophagy induction by 3MA further increased the level of Abeta(42) produced under starvation in HUVECs. These results suggest that starvation-induced Abeta(42) secretion might contribute to the formation of CAA and hence vascular degeneration in AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide , Animales , Vasos Sanguíneos/metabolismo , Encéfalo/metabolismo , Angiopatía Amiloide Cerebral/metabolismo , Células Endoteliales/metabolismo , Humanos , Ratones , Ratones Transgénicos , Nexinas de Proteasas , Receptores de Superficie Celular , Inanición/metabolismo , Venas Umbilicales/metabolismo , OmbligoRESUMEN
Astrocytes play a diverse role in central nervous system (CNS) injury. Production of the serine protease inhibitors (serpins) plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1) by astrocytes may counterbalance excessive serine protease activity associated with CNS pathologies such as ischemic stroke. Knowledge regarding the regulation of these genes in the brain is limited, so the objective of the present study was to characterize the effects of injury-related factors on serpin expression in human astrocytes. Native human astrocytes were exposed to hypoxia or cytokines, including interleukin-6 (IL-6), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), IL-10, transforming growth factor-alpha (TGF-alpha), and TGF-beta for 0-20 hr. Serpin mRNA expression and protein secretion were determined by real-time RT-PCR and ELISA, respectively. Localization of PAI-1 and PN-1 in human brain tissue was examined by immunohistochemistry. Hypoxia and all assayed cytokines induced a significant increase in PAI-1 expression, whereas prolonged treatment with IL-1beta or TNF-alpha resulted in a significant down-regulation. The most pronounced induction of both PAI-1 and PN-1 was observed following early treatment with TGF-alpha. In contrast to PAI-1, the PN-1 gene did not respond to hypoxia. Positive immunoreactivity for PAI-1 in human brain tissue was demonstrated in reactive astrocytes within gliotic areas of temporal cortex. We show here that human astrocytes express PAI-1 and PN-1 and demonstrate that this astrocytic expression is regulated in a dynamic manner by injury-related factors.
Asunto(s)
Precursor de Proteína beta-Amiloide/biosíntesis , Astrocitos/metabolismo , Lesiones Encefálicas/metabolismo , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Receptores de Superficie Celular/biosíntesis , Química Encefálica , Lesiones Encefálicas/patología , Hipoxia de la Célula , Células Cultivadas , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/genética , Humanos , Hipoxia Encefálica/patología , Inmunohistoquímica , Nexinas de Proteasas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Serpina E2 , Serpinas/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.
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Amiodarona/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Bepridil/farmacología , Inhibidores Enzimáticos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Amiodarona/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Bepridil/química , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/metabolismo , Línea Celular , Células Cultivadas , Inhibidores Enzimáticos/química , Femenino , Cobayas , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Nexinas de Proteasas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5'-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2'-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation.
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Regiones no Traducidas 5' , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Proteína 1 Reguladora de Hierro/metabolismo , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Receptores de Superficie Celular/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/patología , Línea Celular Tumoral , Humanos , Hierro/metabolismo , Proteína 1 Reguladora de Hierro/genética , Macaca mulatta , Ratones , Nexinas de Proteasas , Receptores de Superficie Celular/genéticaRESUMEN
RATIONALE: Several family-based studies have identified genetic linkage for lung function and airflow obstruction to chromosome 2q. OBJECTIVES: We hypothesized that merging results of high-resolution single nucleotide polymorphism (SNP) mapping in four separate populations would lead to the identification of chronic obstructive pulmonary disease (COPD) susceptibility genes on chromosome 2q. METHODS: Within the chromosome 2q linkage region, 2,843 SNPs were genotyped in 806 COPD cases and 779 control subjects from Norway, and 2,484 SNPs were genotyped in 309 patients with severe COPD from the National Emphysema Treatment Trial and 330 community control subjects. Significant associations from the combined results across the two case-control studies were followed up in 1,839 individuals from 603 families from the International COPD Genetics Network (ICGN) and in 949 individuals from 127 families in the Boston Early-Onset COPD Study. MEASUREMENTS AND MAIN RESULTS: Merging the results of the two case-control analyses, 14 of the 790 overlapping SNPs had a combined P < 0.01. Two of these 14 SNPs were consistently associated with COPD in the ICGN families. The association with one SNP, located in the gene XRCC5, was replicated in the Boston Early-Onset COPD Study, with a combined P = 2.51 x 10(-5) across the four studies, which remains significant when adjusted for multiple testing (P = 0.02). Genotype imputation confirmed the association with SNPs in XRCC5. CONCLUSIONS: By combining data from COPD genetic association studies conducted in four independent patient samples, we have identified XRCC5, an ATP-dependent DNA helicase, as a potential COPD susceptibility gene.
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Cromosomas Humanos Par 2 , ADN Helicasas/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Edad de Inicio , Anciano , Precursor de Proteína beta-Amiloide/genética , Estudios de Casos y Controles , Mapeo Cromosómico , Femenino , Ligamiento Genético , Predisposición Genética a la Enfermedad , Humanos , Autoantígeno Ku , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Nexinas de Proteasas , Receptores de Superficie Celular/genética , Fumar/efectos adversosRESUMEN
The amphipathicity of the natively unstructured amyloid-beta (Abeta40) peptide may play an important role in its aggregation into beta-sheet rich fibrils, which is linked to the pathogenesis of Alzheimer's disease. Using the air/subphase interface as a model interface, we characterized Abeta's surface activity and its conformation, assembly, and morphology at the interface. Abeta readily adsorbed to the air/subphase interface to form a 20 A thick film and showed a critical micelle concentration of approximately 120 nM. Abeta adsorbed at the air/subphase exhibited in-plane ordering that gave rise to Bragg peaks in grazing-incidence x-ray diffraction measurements. Analysis of the peaks showed that the air/subphase interface induced Abeta to fold into a beta-sheet conformation and to self-assemble into approximately 100 A-sized ordered clusters. The formation of these clusters at the air/subphase interface was not affected by pH, salts, or the presence of sucrose or urea, which are known to stabilize or denature native proteins, suggesting that interface-driven Abeta misfolding and assembly are strongly favored. Furthermore, Abeta at the interface seeded the growth of fibrils in the bulk with a distinct morphology compared to those formed by homogeneous nucleation. Our results indicate that interface-induced Abeta misfolding may serve as a heterogeneous, nucleation-controlled aggregation mechanism for Abeta fibrillogenesis in vivo.